bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunologic Approach to the Study of Factor VIII
Authors: Hoyer Leon W.
Published: May 2019

In contrast to direct biochemical analyses, immunologic studies of factor VIII are less limited by that factor’s lability and low protein concentration. Both human and heterologous antifactor VIII have been used in these studies. Human antibodies inactivate factor VIII procoagulant. activity but they do not fix complement or form immunoprecipitates. Their interaction appears to involve the formation of an immune complex with a univalent component distinct from that which has been designated factor VIII-related protein. This component is synthesized—but remains nonfunctional—in hemophilia, and at least two different defects have been distinguished. Very different reactions can be identified using heterologous antibodies. Although many of these antibodies do not react directly with the procoagulant site, they do fix complement, precipitate with factor VIII-related protein, and inactivate ristocetin cofactor activity. These antibodies are useful for classification of factor VIII-deficiency diseases according to the relative levels of factor VIII procoagulant activity and factor VIII-related antigen; identification of the hemophilic carrier state; characterization of heterogeneity in factor VIII structure; and purification of factor VIII procoagulant activity by immunoadsorbent chromatography. In general, the information obtained by immunologic studies has complemented that from direct biochemical analyses. Both approaches contribute to our evolving understanding of factor VIII structure and function.
Factor VIII Antibodies in Von Willebrand’s Disease
Authors: Mannucci P. M. , Ciavarella N.
Published: May 2019

Precipitating antibodies (Ab) are extremely rare in congenital coagulation disorders and were not described in FVIII deficiency states. Plasma and serum of 4 multitransfused patients (pts) with severe recessive VWD were shown to react in precipitation systems with the FVIII-related antigen (VIIIR:AG) recognized by heterologous Ab. Such property was related to the IgG fraction. Plasma or IgG fractions cross reacted with plasma from a number of mammalians, producing precipition lines with partial identity with human plasma and semipurified FVIII. They also inactivate in a time-indipendent manner FVIII-related ristocetin cofactor (VIIIR:RCo) and inhibit platelet aggregation induced in PRP by bovine and porcine FVIII. FVIII coagulant activity (VIII:C) was inactivated by the Ab and there is evidence that this phenomenon is related to steric hindrance. Transfusion studies carried out in pts during treatment for bleeding episodes showed an impaired in vivo recovery of infused VIIIR :AG and VIIIR :RCo, and an anamnestic rise of Ab;the delayed rise of VIII :C which is usually observed in WD patients was not shown. The findings of precipitating Ab suggest that pts with severe VWD with complete failure to synthetise FVIII have no immunological tolerance to the protein, Challange with a totally foreign protein induced by transfusion is followed by the development of precipitating Ab, This view is supported by the observation that the properties of such homologous Ab are similar to those of the heterologous Ab raised in animals against human FVIII.
Acquired Inhibitors of Factor V
Authors: Feinstein Donald I.
Published: May 2019

Twelve patients with an acquired inhibitor of Factor V have been reported thus far in the literature. Of these, only one occurred in a patient with hereditary Factor V deficiency. Six patients received streptomycin in close temporal relationship to the appearance of the inhibitor. Six of the patients had been previously transfused, including four of those who received streptomycin. In nine of the eleven spontaneously occurring inhibitors, major surgery preceded the appearance of the inhibitor. The degree of clinical bleeding in these patients varied. One patient had no bleeding, six patients had mild to moderate bleeding, and four patients had severe bleeding. The inhibitor disappeared in less than eight weeks in seven patients, whereas in one patient it persisted for more than two years. Most of these inhibitors have the physiochemical properties of antibodies. Six of the spontaneous inhibitors appeared to be IgG, whereas in two patients inhibitor activity was found in both IgM and IgG fractions. Three inhibitors have been typed with light chain antisera and all contained both kappa and lambda chains. Plasmas from seven patients with hereditary Factor V deficiency have been tested with three of these inhibitors for inactive factor V antigenic material and none has been detected. In addition, plasma from a patient with hereditary factor V deficiency has been tested with heterologous factor V antibody and no antigenic material has been found. Thus hereditary factor V deficiency probably represents a deficiency of factor V molecules, rather than the synthesis of a defective molecule.
Hageman Factor (HF) Dependent Fibrinolysis
Authors: Kaplan Allen P. , Mandle Robert , Yecies Lewis D. , Meier Henry L.
Published: May 2019

The HF dependent fibrinolytic pathway is initiated by binding of HF (M. W. 80,000) and a complex of prekallikrein and high molecular weight (HMW) kininogen (M. W. 280,000) to negatively charged surfaces. A reciprocal reaction proceeds in which HFA converts prekallikrein to kallikrein and kallikrein activates HF. The rate of each enzymatic reaction is augmented by HMW kininogen. The active site of HF, as assessed by incorporation of 3H-DFP, in the surface bound enzyme does not form upon binding in the presence or absence of HMW kininogen, but is generated upon activation by kallikrein. The product, HFA (M. W. 80,000), is subsequently cleaved to liberate the active Hageman factor fragments (M. W. 28,000). Two forms of prekallikrein (M. W. 88,000 and 85,000) are cleaved by HFA to yield kallikreins in which a heavy chain (M. W. 52,000) is disulfide linked to a light chain (36,000 or 33,000) and, for each molecular form, the active site is in the light chain. Kallikrein activates plasminogen (M. W. 94,000) to yield a plasmin consisting of a heavy chain (M. W. 58,000) disulfide linked to a light chain (M. W. 27,000) and again, the active site is in the light chain. Digestion of prekallikrein by kallikrein yields proenzymes of molecular weight 78,000 and 75,000 that appear to represent the previously described plasminogen proactivator. Factor XI circulates bound to HMW kininogen, is activated by HFA in the presence of HMW kininogen, and factor XIA-HMW kininogen activates HF. Thus factor XI may contribute to the gradual activation of HF and evolution of fibrinolytic activity in prekallikrein deficient plasma. A further role for factor XIA as a plasminogen activator will be discussed.
Antiplasmin, the Fast-Reacting Plasmin Inhibitor of Human Plasma
Authors: Collen D. , Wiman B.
Published: May 2019

Recently a new plasmin inhibitor was discovered in human plasma by three different groups (Eur. J. Biochem. 69, 209, 1976; J. Biol. Chem. 251, 5956, 1976; Biochem. J. 159, 545, 1976). This inhibitor is responsible for the fast-reacting plasmin-neutraIizing activity of human plasma and forms a very stable 1:1 molar complex with plasmin, which is devoid of protease and esterase activity. The concentration of antiplasmin in human plasma is approximately I μM (2/3 that of plasminogen).

We have purified the inhibitor by affinity chromatography on insolubilized plasminogen, followed by DEAE-Sephadex and concanavalin A chromatography. It is a glycoprotein with a mol . wt. of 67,000 (SDS-gel electrophoresis), a carbohydrate content of 14%, and amino terminal sequence Asn-Gln-Glu-Gly (Wiman and Collen, submitted).

The inhibitor interacts strongly with the light chain of plasmin (probably through reaction with the active site serine), and weakly with the heavy chain (probably through interaction with the lysine-binding site), suggesting that antiplasmin not only functions as an inhibitor of plasmin, but also interferes with the interaction between plasm in (ogen) and fibrin.
In Vivo Studies with a New Antiaggregating Drug EMD 26644, A Mercapto-Oxazolyl-Carbon-Acid Derivative
Authors: Krzywanek H. J. , Pietsch U. , Breddin K.
Published: May 2019

The inhibitory effect of EMD 26644 on platelet function has been studied with the following methods: spontaneous aggregation (PAT III), ADP-, collagen-and epinephrire-induced aggregation, primary shape change of thrombocytes at 37°C incubation temperature and platelet spreading.

Duke’s bleeding time, platelet count and some coagulation parameters like partial thromboplastin time, Quick’s test, fibrinogen, thrombin time and factor VII-assay as well as platelet spreading were unchanged throughout the test period.

An aspirin like inhibitory effect on platelet aggregation and primary shape change was observed 48 – 72 hrs. after the ingestion of a single dose of 250 mg EMD, which lasted for 2–3 weeks. A prolonged antiaggregating effect was achieved by the continuous oral administration of 25 – 50 mg EMD for a period of 2 – 3 months. No potentiation of the antiaggregating effect was seen, if ASA was given in addition to EMD 26644.

In comparison with aspirin the new compound seems to have some advantages: the same antiplatelet properties are achieved with a much lower dose and no side effects have been reported so far. Clinical studies will have to confirm whether EMD 26644 is an effective antithrombotic agent.
Alterations in The Trans-Membrane Potential of Human Blood Platelets in Response to Aggregating Stimuliow
Authors: Horne W. C. , Larsen N. E. , Simons E. R.
Published: May 2019

It has been suggested that the mechanism of response of platelets to aggregating stimuli is analogous to the stimulus-response coupling mechanisms of muscle and secretory cells. Because the coupling mechanisms of both types of systems involve changes in the trans-membrane potential of the stimulated cell, we have examined the membrane potential of human platelets to determine if the potential changes in response to aggregating agents. The membrane potential of gel-filtered platelets was monitored using a fluorescent cyanine dye. Aggregation was prevented to minimize artifacts due to changes in light scattering. The effects of ADP, thrombin, and collagen on the potential were examined. In response to ADP, the membrane rapidly became more negatively polarized. The response was concentration dependent, the minimum response occurring with 3x10-5M ADP. The response to thrombin was more complex. Low concentrations (0.01 U/ml) produced a change similar to that caused by ADP. Higher concentration (1–10 U/ml) led to a rapid decrease in the polarization of the membrane. When pre-formed collagen fibrils were added, in the presence of creatine Phosphokinase to destroy released ADP, no changein the potential was observed.

Thus the platelet membrane potential changes in response to aggregating stimuli, supporting the hypothesis that stimulus of platelet aggregation is analogous to other stimulus-response coupling mechanisms. The change in potential is not a necessary step in the coupling mechanism. The different responses to specific agents indicate that the agents may trigger aggregation through different mechanisms.
von Willebrand Factor Activity in Plasmin Digests of Factor VIII
Authors: Guisasola J. A. , Cockburn C. , Hardisty R. M.
Published: May 2019

Purified human factor VIII was incubated for up to 24 hours with plasmin, and the activity of the breakdown products studied at intervals. Factor VIII coagulant activity was lost within the first hour, but von Willebrand factor activity (FVIIIR:WF) was retained for two hours, and then declined slowly during the subsequent incubation. Analysis of the 24-hour breakdown products by immuno-electrophoresis, sepharose 4B chromatography and SDS Polyacrylamide electrophoresis revealed three main groups of fragments recognised by rabbit anti-human factor VIII anti-serum, and having molecular weights in the following ranges: Group 1 300,000=500,000; Group II, 150–200,000; Group III, 100,000. FVIIIR:WF activity, which was found only in Group II, appeared to be associated with glycopeptide(s) of up to 155,000 daltons.
In Vivo Fibrinopeptide a Generation Induced by Angiographic Catheters
Authors: Wilner G. D. , Casarella W. J. , Fenoglio C. , Baier R. E.
Published: May 2019

The use of fibrinopeptide A (FPA) levels to assess the hemostatic biocompatibility of prosthetic devices has been evaluated. Commercial angiographic catheters (7 French) of 5 different materials were introduced percutaneously 10 cm retrograde into the femoral arteries of anesthetized mongrel dogs. Catheter lumens were continually flushed with saline (1 ml/min). At intervals over a 30 minute period, saline flow was stopped and blood samples were withdrawn through the catheter lumen for FPA assay. Clot formation in catheter lumens was evaluated by scanning electron microscopy (SEM). The catheters could be divided into three groups based on FPA level generated and deposition of fibrin and platelets in the lumen. Group I (the PERT catheter) caused no increase in FPA (mean level 0.5 pmol/ml, equal to the level in the absence of any catheter), no fibrin deposition, and moderate platelet deposition. Group II (Torcon and Teflon catheters) caused moderate FPA generation (mean 7.1 pmol/ml), moderate fibrin deposition, and moderate platelet deposition. Group III catheters (polyurethane and Dacron) caused marked FPA generation (mean 26.2 pmol/ml), marked fibrin deposition, but variable platelet deposition. Thus mean FPA levels over the 30 minute experimental period correlated well with intraluminal fibrin deposition; FPA measurements should be useful in assessing the degree to which different catheters stimulate fibrin formation. FPA levels did not correlate with intraluminal platelet deposition and thus will not reflect platelet contributions to thrombus formation.
Changes in Levels of Plasma Pre-Kallikrein, Kallikrein and Kallikrein Inhibitors During Endotoxin Shock in Dogs
Authors: Gallimore M. J. , Aasen A. O. , Amundsen E.
Published: May 2019

Plasma protease activity is known to be increased during endotoxin shock and recent studies have indicated that the plasma kallikrein-kinin system becomes activated by circulating endotoxin. Plasma levels of pre-kallikrein kallikrein and kallikrein inhibitors were therefore determined in samples from dogs infused with E. coli endotoxin, using assays with a chromogenic substrate for plasma kallikrein(Chromozyme -PK, Pentapharm, Basle, Switzerland). “Fast-reacting” and “time-dependent” inhibitors of kallikrein were studied using purified human plasma kallikrein. Considerably reduced levels of plasma pre-kallikreiri and increased levels of kallikrein were detected in the late phase of shock and significant reductions in “fast-reacting” and “time-dependent” inhibition of kallikrein was observed. These results show that during endotoxin shock plasma pre-kallikrein becomes activated to kallikrein and indicate that kallikrein inhibitors play an important mediatory role in the pathophysiology of endotoxin shock.
Studies on a Novel Circulating Anticoagulant in a Female with Bleeding Diathesis
Authors: Messmore Harry L. , Fareed J. , Chang Roberta L. , Gawlik Gerald M. , Kozuh Gerald F.
Published: May 2019

A 42 year old Caucasian female has had a history of bleeding from surgical trauma since the age of 3. She has bled massively from surgical trauma and has had life threatening postpartum hemorrhages. Laboratory tests showed marked prolongation of thrombin time, prothrombin time, partial thromboplastin time, low α1 antitrypsin and lipoprotein lipase activity. Fibrinogen level was normal. Mixing of patient’s plasma and serum with citrated normal plasma suggested the presence of a potent antithrombin (AT) substance. Heating of both the plasma and serum at 56°C for 2 hours failed to destroy the AT activity. The AT activity of the patient’s plasma was quantitated to be equivalent to 0.75–0.9 u/ml heparin. Platelets aggregated normally with ADP, collagen, epinephrine, but not with Ristocetin and thrombin (0.5–5.0 u/ml). Her plasma and serum blocked thrombin induced aggregation of normal platelets. Her plasma also inhibited the factor Xa induced hydrolysis of S-2222 (Bz-Ile-Glu-[γ-OR]-Gly-Arg-pNA) and the action of thrombin on S-2160 (Bz-Phe-val-Arg-pNA). The inhibitor was not adsorbed with barium sulfate or reduced in activity by protamine sulfate, toluidine blue or heparinase. It was chromatographed on Sephadex G-100 and G-50, and eluted in the void volume and a low molecular (10–20,000) fraction. Incubation of the patient’s plasma with chymotrypsin and trypsin destroyed the anticoagulant activity. Although anti-AT-III serum totally neutralized the anticoagulant activity of heparin-ized plasma, it failed to neutralize the patient’s plasma anticoagulant activity. Albumin partially neutralized the anticoagulant activity. These studies suggest that this AT substance is a potent inhibitor of serine proteases and behaves as activated AT-III.
Inactivation of Thrombin and Factor Xa by Antithrombin III and Plasma: Studies with Bovine and Human Enzymes
Authors: Ødegård O. R. , Abildgaard U.
Published: May 2019

The results of some earlier studies have indicated that At-III preferentially reacts with Xa rather than thrombin (1) . We have estimated the inactivation rates using chroraogenic substrates (2,3) to measure remaining enzyme activities after incubation with plasma or purified At-III. The enzyme half-time values were approximately inversely proportional to the At-III or plasma concentration. When added to purified human At-III or plasma, human Xa was more slowly inactivated than human thrombin. With an At-III activity of 70% of normal,the half-time of human thrombin was about 96 seconds,of human Xa about 150 seconds. Simultaneous incubation of At-III with human thrombin and Xa confirmed that thrombin was preferentially inactivated. Purified human At-III behaved similar to human plasma. The results indicate that with human reagents, At-III preferentially reacts with thrombin rather than with Xa. The enzymes of bovine origin were more rapidly inactivated than the human enzymes. Heparin accelerated the inactivation rates, but did not alter the order of inactivation rates between the enzymes.
Familial Thrombosis Due to Antithrombin III Deficiency: An Extensive Family Study
Authors: Bennett Bruce , Mackie Michael , Douglas Alexander S.
Published: May 2019

A family living in Lewis (a Hebridean Island off the north west coast of Scotland) affected by antithrombin III deficiency has been studied. Two members have died, one of massive pulmonary embolism and one of major mesenteric infarction secondary to mesenteric vein occlusion. A further individual has sustained major small bowel infarction secondary to mesenteric vein thrombosis but survived after two small bowel resections followed by anticoagulant therapy. Other members of the family have suffered from nonfatal thrombotic events particularly during pregnancy. 57 individuals representing several generations have been studied and will be presented. Levels of antithrombin III measured by functional assays correlated well with those of antithrombin III measured immunologically. 12 members of the family showed moderate to severe deficiency of antithrombin III, the occurrence of thrombotic symptoms correlating well with deficiency of this protein. Transmission of the disorder as an autosomal dominant disorder is confirmed in the patients studied and by historical evidence over many generations.
Platelet-Collagen Interaction : Adhesion to Alpha 1 (I) CB6 and Alpha 1 (III) CB4 Peptides from Calf-Skin Collagen
Authors: Fauvel F. , Legrand Y. J. , Caen J. P.
Published: May 2019

The interaction of blood platelets with collagen is the trigger of haemostasis and, at least partly, of thrombosis. Two types of fibrillar collagens (type I and type III), present in the vessel wall, can induce the adhesion of blood platelets. This study was designed to determine which part of their molecule is involved in that phenomenon. We then devised a quantitative test based upon differential filtration of non adhesive and adhesive 14c serotonin labeled platelets through a sepharose column for the evaluation of platelet adhesion to collagen or collagen derivatives. No differences were observed in the adhesive properties of calf-skin type I and type III collagens when the degree of multimerisation was identical. Isolated alpha 1 (I) and alpha 2 chains were not active ; but platelets adhered to reassociated (alpha .1 (I))3 trimers, not to (alpha 2)3 trimers ; in type I collagen, alpha 1 chains seems to have the most essential role for platelet adhesion. Various cyanogen bromide peptides were tested : only the 217 C-terminal amino acids alpha 1 CB6 from type I and the 149 central amino acids alpha 1 CB4 from type III were active.

The adhesive properties of collagen seems then associated with chemical groupings localized in these peptides : a random folding of the entire isolated chains could make these “sites” unavailable to the platelets, whereas in the triple helix characteristic of fibrillar trimers, they would be more accessible. This could explain why fibrils and small peptides are active, while isolated chains are not.
Characterization of the Binding of Adenosine Diphosphate to Human Platelet Membranes
Authors: Legrand C. , Bauvois B. , Caen J. P.
Published: May 2019

ADP-mediated platelet aggregation is a routinely employed test but its mechanism is poorly understood. The aim of this study was to compare the binding of ADP to plasma membranes isolated from normal platelets and thrombasthenic platelets (which do not aggregate with ADP). Binding of ADP to isolated membranes was assayed by incubation with 14C-ADP followed by Mill i pore filtration. In standard conditions, 14C-ADP was not transformed and non specific binding represented lessthan 3 % of the total binding. Using 1 μM 14C-ADP, the binding has been shown to be a rapid (t 1/2 = 2 mn 30 sec), saturable and reversible phenomenon at 37° C. The existence of a major population of binding sites, with an affinity constant Ka = 0.43 (+ 0.1) χ 106M-1, has been demonstrated. The kinetics of the binding was normal with membranes Tsolated from the platelets of 4 thrombasthenic patients and the affinity constant, when determined, was in the normal range. Dissociation of the membrane-bound 14C-ADP occurred rapidly at 37° C (t l/2c≃3mn) when samples were diluted enough (dilution 1 : 100 was currently employed) to avoid rebinding of the radioligand. Accelerated dissociation (t 1/2 ≃ 1 mn) was observed when the dilution was performed in the presence of an excess of unlabeled ADP, suggesting the existence of negatively cooperative site-site interactions among the ADP binding sites. This effect was only observed at high concentrations of ADP (> 10–5M) and its eventual role in vivo remains to be established. Two thrombasthenic membrane preparations studied in the same way dissociated as did the control membranes.
ADP Binding Proteins in Thrombasthenic Platelet Membranes
Authors: Bennett Joel S. , Friedman Schlomo , Colman Roberta F. , Colman Robert W.
Published: May 2019

Thrombasthenia is a severe congenital platelet disorder characterized by absence of ADP-induced platelet aggregation, diminished clot retraction and abnormalities of platelet membrane glycoproteins. The finding of normal ADP-induced shape change suggests that initial ADP binding might be normal. We have previously shown that the synthetic ADP analogue 5′-p-fluorosul-fonylbenzoyladenosine (5′FSBA) inhibits ADP-induced platelet shape change and covalently binds to specific proteins in normal platelet membranes. This affinity label was thus used to identify ADP binding proteins in platelet membranes from a patient with thrombasthenia. Isolated platelet membranes were incubated with 3H-5′-FSBA (0.1μM) at 37°C for 1 hr. then dissolved in 19% SDS, 8M urea, and 0.2M dithiothreitol, and subjected to SDS disc gel electrophoresis. The radiochromatograms of disc gels from 16 normal individuals demonstrated consistent labelling of polypeptides of 200, 120, 100, and 43 χ 103 daltons. The covalent binding was almost completely prevented by simultaneous incubation with a 100-fold excess of ADP or ATP, only partially prevented by AMP, adenosine and GDP, and was unaffected by UDP, thrombin, and epinephrine. When 5′FSBA was incubated with membranes from thrombasthenic platelets, the incorporation was identical to that found in normal membranes. Further the SDS-disc gel pattern showed the same 4 radiolabelled polypeptides despite the characteristic thrombasthenic membrane protein abnormalities. These experiments demonstrate that the ADP binding proteins of thrombasthenic platelet membranes are similar to those of normals and support the concept that the defect in thrombasthenia occurs subsequent to initial ADP binding.
Heparin Elimination in Patients with Various Diseases
Authors: Teien A. N. , Lie M.
Published: May 2019

Heparin (100 U/kg bodyweight) has been administered as i. v. single injections and heparin concentration in plasma determined by polybrene titration (PT), previously shown to be an accurate assay for heparin concentration in plasma. Mean concentration half-life was 74.7 min. in normals (n=6),118.6 min. in a-nephric patients (n=5), 97.8 min. in uraemic patients with kidneys (n=6), 115.5 min. in patients with liver cirrhosis (n=6),79.6 min. in patients with disseminated cancer (n=5) and 59.3 min. in patients with hyper-lipaemia(n=6). The differences between mean values for the normals and the anephric patients or the patients with liver cirrhosis were highly significant (p<0.01). One patient with very high content of triglycerides in plasma (25.6 mmol/l) had an extremely short heparin half-life (33 min.). The plasma of this patient had a heparin neutralizing effect of 0.75 U/ml (measured by FT) . Short half-lives were also found in patients with thrombocytaemia or acute thrombo-embolic disorders.

In normals and uraemic patients there was a significant fall in heparin co-factor activity from before heparin injection to 4 hours after injection (lO per cent and 20 per cent, p<0,05 and p<0,005»respectively),in contrast to no significant fall in patients with liver cirrhosis, A possible explanation may be that the normal liver removes heparin bound to At-III, and that this function is impaired in liver cirrhosis.
Analysis of Soluble Fibrin Complexes in Patients with Intra Vascular Coagulation
Authors: Edgar W. , McKillop C. , Howie P. W. , Forbes C. D. , Prentice C. R. M.
Published: May 2019

We have analysed the polypeptide chain structure of soluble fibrin complexes in patients with pre-eclampsia and during defibrination with ancrod (Arvin). In pre-eclampsia soluble fibrin measured by fibrinogen-sepharose 4B chromatography was increased in comparison with normal pregnant women. The isolated fibrin contained intact α, β and δ chains but no crosslihked γ chains. A correlation (r = 0.959 P <0.001) was found between the concentration of soluble fibrin and soluble complexes as measured by agarose gel filtration, suggesting these complexes consist mainly of fibrin-fibrinogen dimers. In vitro, the binding of soluble fibrin to fibrinogen-sepharose depended on the structure of the complexes and temperature. At 37°C intact fibrin, prepared with ancrod, was bound to the column, but fibrin lacking intact α chain could be eluted at 37°C. The soluble fibrin complexes in patients having therapy with ancrod could be separated into two;the greater part had reduced α chain and were eluted from the column at 3 7°C, whereas the smaller part had intact α chain and remained bound at 37°C. Bio-Gel A5m chromatography carried out at 37°C and 20°C also indicated that the major part of soluble complexes produced during ancrod defibrination were unstable at 37°C.
Changes in Distribution of Platelet Membrane Glycoproteins in Patients with Myeloproliferative Disorders
Authors: Bolin R. B. , Okumuru T. , Jamieson G. A.
Published: May 2019

A tendency towards hemorrhage can occur in patients with myeloproliferative diseases and in many patients abnormal platelet function can be detected by various techniques. Platelet aggregation studies have shown decreased response to aggregating agents and a few patients may have no response to ADP, collagen and epinephrine and thus may mimic the platelet defect seen in patients with thrombasthenia. We have studied membrane glycoprotein patterns in 16 patients with various myeloproliferative disorders and compared the results to normal controls since glycoproteins have been implicated as being important to platelet function both in normal and pathological states. Although no abnormal glycoproteins were present, there was an abnormal ratio compared to the controls with a relative decrease in glycoprotein I (M. W. 150,000) and an increase in glycoprotein TV (M. W. 87,000) that was statistically significant (α0.005) . There was no discernible correlation between glycoprotein pattern and aggregation response or platelet count but those patients with megathrombocytes did have higher values for glycoprotein IV than those without megathrombocytes. These experiments suggest that patients with myeloproliferative disorders may have alterations in membrane glycoproteins that could alter platelet function.
Inhibition of Platelet Aggregation by Brinolase Degradation Products of Fibrinogen and Serum Albumin
Authors: Roschlau W. H. E.
Published: May 2019

Brinolase (fibrinolytic enzyme from Aspergillus oryzae) was observed to possess significant platelet aggregation inhibitory properties during and after thrombolytic therapeutic use. These platelet effects were found in vitro to be caused in part by intermediate products of fibrinogen digestion, namely low-molecular-weight peptides of approx. MW 2500. Human fibrinogen peptides were isolated, purified, and shown to have high inhibitory activity in platelet-rich plasma. Quantitative comparisons of attainable platelet inhibition in vitro and observed responses in vivo during administration of equivalent enzyme doses, however, suggested that total available fibrinogen, even if it were entirely converted to degradation products (which it is not), would be insufficient to account for observed platelet effects of brinolase therapy.

Human serum albumin is also readily degraded by brinolase. Albumin degradation products were prepared in vitro by optimal incubation with the enzyme. Dose-response curves of inhibition of platelet aggregation were obtained with lyophilized peptides in platelet-rich plasma in vitro, and significant inhibition of platelet aggregation was observed in vivo following infusion of albumin degradation products into rabbits. The enzyme doses and amounts of substrates employed in all experiments were equivalent to the conditions of therapeutic fibrinolysis.

Thus, albumin degradation products are considered to contribute a significant, if not the major, portion of platelet-active intermediates during clinical brinolase therapy. Albumin cleavage, which is unique to brinolase amongst clinical fibrinolytic enzymes, was shown to have biological effects of its own, but it may also serve to protect coagulation proteins from enzymatic destruction through competition for the enzyme during systemic brinolase therapy.
Blood Product Infusions to Maintain Normal Haemostasis Parameters in Low Birth Weight Neonates
Authors: Cash J. D. , Turner T. L.
Published: May 2019

Haemostatic parameters were measured serially for 3 days in 58 infants, within 2 hours of birth. Twelve (21%) maintained normal coagulation profiles, whereas 46 (79%) had abnormal profiles. The abnormal group were randomly allocated to a control (23) and treatment subgroup (23).Treatment consisted of infusions of platelets, fibrinogen (cryoprecipitate) and/or factor II, VII, IX & X concentrate – whichever was appropriate, and repeated when necessary. Detailed post-mortem studies were performed on all infants who died.

The results revealed a lower incidence of intraventricular haemorrhage in the treated subgroup, but this was not statistically significant. There was no difference in mortality between the two subgroups. No deleterious effects due to the administration of blood products;in particular, DIC or local thrombosis was not observed.

It is concluded that although efforts to maintain normal haemostasis in these high risk neonates are possible and may be safe, they are without demonstrable benefit.
Template Bleeding Time:Influence of Techniques
Authors: Mielke C. H. , Flavell P. , Rodvien R.
Published: May 2019

We have studied how specific changes in technique affects the bleeding time (BT). Using a template BT (TBT), we have evaluated the influence of a blood pressure cuff set at 40 mm. Hg. The cuff influenced the TBT, and increased the sensitivity to aspirin(ASA)(N = 12,

= 5′00″ to 10′16″ versus 2′41′ to 5′08″, p < .05). A vertical incision produced a shorter TBT than a horizontal incision (N = 100,

= 4′21″ versus 5′34″, p < .001). In 9 normal subjects, the Ivy(I) and the Duke (D) methods were compared to vertical (V) and horizontal (H) TBTs with and without ASA. D was not influenced by ASA, and I showed a mild prolongation which returned to normal in 3 days. The TBT showed the greatest prolongation (H > V) returning to normal within 6 days. The TBT was then compared to a Warner-Lambert (WL) spring loaded device which produced a 6 χ 1 mm vertical incision. Eight subjects were studied twice weekly for 5 weeks. The

of TBT was shorter than the WL (N = 80,

– 4′14″ + 4′14″ versus 5′28″ + 1′54″) and the range was smaller. Neither technique was painful and scarring was minimal. The repeatability and sensitivity of the BT to ASA is highly dependent on the technique used. Either the BT measures more than platelet participation in hemostasis or different BT techniques are variably sensitive to different pathways of platelet recruitment.
Platelet Survival Using Aspirin: Studies in Patients with Artificial Cardiac Valves
Authors: Goodnight S. H. , Montgomery L. M. , Sheth K. , Pipoly G. M.
Published: May 2019

Studies using 51Cr-labeled platelets have shown reduced platelet survival in patients with artificial cardiac ball valves. Recently, a simple non-radioisotopic method for measuring platelet survival has been developed by Stuart (NEJM 292:1310, 1975) based on the concept that aspirin (ASA) irreversibly inhibits lipid peroxidation of circulating platelets. This method was used to determine platelet survival in 20 control subjects, 8 patients with normally functioning Starr-Edwards artificial cardiac valves (cardiac catheterization 5/8) and 4 patients with documented prosthetic valve dysfunction. The T½ (best fit to an exponential curve) for control subjects was 4.0 ± 0.7 (1 SD) days with a range of 3.0 to 5.6 days. The T½ of normally functioning artificial valves was less than that of the control subjects (3.3 ± 0.3 days, range 3.1 – 3.8 days, p < 0.05). Moreover, the patients with valves that were obstructed or had a peri prosthetic leak had a T½ of 2.7 ± 0.1 days, which was significantly shorter than either the controls or the patients with normally functioning artificial valves (p < 0.01). Two additional valve patients also showed shortened platelet survival (subacute bacterial endocarditis – T½ 2.9 days, acute intimai tear of the aorta – T½ 2.5 days) and 2 patients without prostheses who were taking dipyridamole had normal platelet survival [T½ 4.0, 3.3 days). These results indicate that the ASA method for determining platelet survival identifies shortened platelet survival in patients with artificial cardiac valves and may be a useful non-invasive tool to detect prosthetic valve dysfunction.
Another Inherited Abnormality in Plasma Fibrinogen
Authors: Bosch N. B. , Arocha-Piñango C. L. , Rodriguez A. , Ojeda A. , Carvajal Z.
Published: May 2019

A prolonged thrombin and reptilase time with normal fibrinogen concentration in a 9 — years old girl, with no history of bleeding, prompted us to study the behaviour of her fibrinogen. Thrombin and reptilase time in different conditions, immunolectrophoresis, immunodiffusion and fibrin polymerization were performed. The thrombin time was partially -corrected by calcium, ionic strength and increasing concentration of thrombin. Fibrin polymerization rate and monomer aggregation time were moderately abnormal. Immunodiffusion and immunoelectrophoresis showed lines of identity with the normal. The electrophoretic -mobility of the α β and γ fibrin chains was normal, but there was a large amount of α — chain left in the cross-linked fibrin,in the presence of factor XIII. The above results suggest the presence of another fibrinogen variant.
Methodological Problems Relating to the Eligibility of Clinical Outcomes in Cardiovascular Intervention Studies
Authors: Gent M.
Published: May 2019

Many patients in long-term cardiovascular clinical trials fail to complete the study according to the original protocol and this raises questions of what should be done with the results from such patients. Examples of protocol failures would include (l) patients dropping out of the trial for reasons which may, or may not, be related to their treatment or clinical condition, (2) interruptions in treatment which may be patient- or physician-initiated, and (3) patients entered into the study who were subsequently found not to meet entry criteria. In practice, the decision to include an observed clinical outcome in the final analysis has usually depended on whether the outcome occurred before or subsequent to the patient leaving the study, and on whether or not the patient was on the allocated treatment at the time of the event.

However, this approach is too simplistic and it can be shown that this will often lead to considerable bias in the evaluation of the treatment under study. The need for more specific decision procedures will be illustrated by examples in previously published trials of anticoagulants, platelet suppressive drugs, and thrombolytic agents.

Recommendations for dealing with these problems will be made which would be pertinent to the several major intervention studies in myocardial infarction and stroke which are currently in progress.
Controlled Trial of Sulfinpyrazone in Rheumatic Heart Disease. Preliminary Results
Authors: Steele Peter , Rainwater Joseph , Genton Edward
Published: May 2019

Platelet survival time (SURV) has correlated with a history of thromboembolism (TE) in patients with rheumatic heart disease (RHD). A controlled trial of sulfinpyrazone (SFP) in RHD is in progress and 138 patients have been entered. SURV (51Chromium labelling) was shortened (2.3 ± 0.08 days; AVE t 1/2 ± SEM; normal 3.7 ± 0.04 days) in 40 of 41 (98%) with a history of TE and in 76 of 97 (78%) (2.9 ± 0.07 days; P<0.001) of those without a history of TE. One hundred sixteen with shortened SURV have been randomized to SFP or placebo and 67 have either completed four years (N=37), undergone mitral valve replacement (N=18) (ave 19 months; range 6-32 months), had definite TE (N=8) (average 14 months; range 8-23 months) or died (N=4) (average 15 months; range 5-22 months). Definite TE (prolonged neurologic deficit) occurred in one on SFP and in seven on placebo (all with shortened SURV) (X2 = 4.31; NS). SFP increased SURV (2.4 ± 0.12 to 2.7 ± 0.13 days; N=23; P<0.001) and 12 (52%) had an increase in SURV of >0.02 days. The patient on SFP with new TE had no change in SURV (2.3 to 2.3 days). Placebo did not alter SURV (2.4 ± 0.15 to 2.5 ± 0.08 days; N=26; NS) and two (8%) had an alteration of SURV by > 0.02 days. SURV was not altered in patients with normal SURV (3.7 ± 0.08 to 3.6 ± 0.08 days; N=12; NS) and no patient with normal SURV has had shortened SURV on subsequent yearly measurement. Patients with normal SURV were not randomized. Results suggest that SURV is shortened in patients with RHD who have had or will have TE, that SFP increases SURV and may prevent TE in these patients.
Depression of Collateral Blood Flow Following Arterial Thrombosis
Authors: Schaub R.G. , Meyers K.M.
Published: May 2019

Permanent ligation of the feline aorta at the iliac bifurcation is followed by rapid opening of pre-existing collateral blood vessels. However, if ligation is combined with formation of a clot these protective collateral vessels do not function. This study was undertaken to determine if drugs which alter serotonin (5-HT) function can improve collateral blood flow after arterial thrombosis. Permanent ligations were placed at the iliac bifurcation, circumflex iliac and sixth lumbar arteries in all cats. Control cats (8) were acutely ligated. In all other cats a clot was produced in the aorta by injection of 0.1 ml of thromboplastin. Clotted cats were untreated (8); had blood 5-HT depleted using a single dose of reserpine (0.1 mg/kg i.m.) followed by para-chlorophenylalanine (p-CPA) (100 mg/kg orally) every 3 days (9); or were treated prior to surgery with a 5-HT antagonist cinanserin HCl (4 mg/kg i.v.) (8). Collateral circulation was determined by blood flow measurements and aortograms 3 days after occlusion of the aorta. The hindlimb blood flow of untreated clotted animals was 20% of the acutely ligated control animals 3 days following aortic occlusion. However, hindlimb blood flow was 90% of control in reserpine and p-CPA treated cats and 60% of control in cinanserin HCl treated cats. Blood flow measurements correlated with aortograms. These results suggest: (1) The clinical consequences of arterial thrombosis cannot be entirely attributed to mechanical occlusion of an artery, but may be due to depression of protective collateral blood flow induced by thrombosis, (2) Serotonin is an important factor in this depression of collateral blood flow, and (3) Isolation of the factors responsible for collateral inhibition could permit the development of therapeutic interventions.
External Pneumatic Compression of the Legs to Prevent Venous Thromboembolism in Neurosurgical Patients
Authors: Collins R. , Coe N. , Goldstein E. , Shapiro R. , Skillman J. , Zervas N. , Salzman E.
Published: May 2019

Neurosurgical patients are at high risk of venous thrombosis (DVT) and pulmonary embolism (PE) but are poor candidates for antithrombotic drugs for fear of intracranial bleeding. Turpie et al. found external pneumatic compression of the legs (EPC) for five days prevented DVT while applied, but 10 days later EPC group and controls were not different. We compared EPC with inflatable boots vs.. untreated controls in 94 patients with trauma or operations of central nervous system or ruptured intracranial aneurysm, continuing EPC throughout period of bedrest (up to 17 days). Diagnosis of DVT was by 125I-fibrinogen scan confirmed by phlebography in cases with positive scans. Results (Table) showed EPC gave significant protection against DVT in neurosurgical patients. There were no complications of EPC. There were no pulmonary emboli, even in the control group, so surveillance by 125I-fibrinogen scan may help to prevent PE.
The Role of Dihydroergotamine in the Prophylaxis of Postoperative Deep Vein Thrombosis
Authors: Kakkar V.V. , Stamatakis J.D. , Lawrence D.
Published: May 2019

In randomly allocated trials, the prophylactic efficacy of DHE and a combination of DHE with heparin was investigated in 200 consecutive patients. Of 100 patients undergoing major surgery, 50 received 0.5 mgm. of DHE every 12 hours and 50 received 50000 units of heparin every 8 hours. The frequency of DVT, determined by the 125I-fibrinogen uptake test, was 20% in those receiving DHE compared with 4% in those receiving heparin, the difference being statistically significant (p 0.05).

Of 100 patients undergoing total hip replacement arthroplasty, 50 received 5000 IU of heparin every 8 hours, and 50 received heparin according to the same regimen, together with 0.5 mgm. of DHE. DVT was detected by the 125I-fibrinogen test and venography. Twenty-six (52%) patients in the heparin group developed DVT, compared with 1– (20%) who received the combination; the difference in the incidence of thrombosis between the two groups was statistically highly significant (p 0.005).

The effect of DHE on calf muscle blood flow was determined using Xenon133. There was significant increase in mean muscle blood flow 2 hours after the administration of 0.5 mgm. of DHE subcutaneously (p 0.05).

It is concluded that a combination of DHE and heparin is more effectively than heparin alone in preventing postoperative DVT.
The Primary Structure of Antithrombin-III
Authors: Petersen T. E. , Dudek-Wojciechowska G. , Sottrup-Jensen L. , Magnusson S.
Published: May 2019

Human antithrombin-III is a single-chain glycoprotein with three disulfide bridges and four prosthetic glucosamine-based oligosaccharide groups. The disulfide bridges have been established. In four fragments of 208, 168, 3 and 46 amino acid residues, resp. 415 of the appr. 425 residues have been sequenced. The four oligosaccharide groups are attached to four Asn-residues within a sequence region of 95 residues. No extensive sequence homology with the trypsin inhibitors has been observed. One chymotryptic peptide was found to be a substrate for bovine factor Xa, cleaving the arginyl bond in the sequence -Ile-Val-Ala-Glu-Gly-Arg-Asp-. A second peptide is cleaved by thrombin. It is not clear whether these sites are inhibitor sites in the native molecule. Other possible candidates for inhibitor sites are a -Val-Leu-Ile-Leu-Pro-Lys-Pro- sequence (similar to the sequence 40-48 of hirudin, which also includes a -Pro-Lys-Pro- sequence) and also the C-terminal sequence -Gly-Arg-Val-Ala-Asn-Pro-Cys-Val-Lys.
The Lipolytic Activity of Antithrombin III
Authors: Nakagawa M. , Okuda S. , Watada M. , Ijichi H.
Published: May 2019

Antithrombin III in the circulating blood is known to play an important role on the coagulation mechanism of the blood and is reported to be decreased in its amount on the cases of myocardial infarction and arteriosclerosis, in the meanwhile the arteriosclerosis has been considered to be one of thrombogenic factors. This research was designed to investigate the effects of antithrombin III on the relationship between fibrin formation and atherosclerosis.

Antithrombin III was purified from human defibrinated plasma by Heparin Sepharose Affinity Chromatography. Using this antithrombin III as an enzyme, its lipolytic activity was assayed spectrophotometrically detecting the produced free fatty acid from various substrates, Intralipid, Triolein, and Human Chylomicron respectively. Lipoprotein lipase activity was detected in the purified antithrombin III. Intralipid was hydrolyzed to a greater extent than triolein or chylomicron and it is concluded that antithrombin III in the circulating blood has not only the inhibitory activity of thrombin but also has the lipolytic activity. From these observations it is considered that this lipolytic activity is beneficial for preventing the developement of atherosclerosis and also of thrombosis.
In Vivo Assessment of Thromboresistant Materials by Determination of Platelet Survival
Authors: Coe N. , Collins R. , Jagoda A. , Brier D. , Lindon J. , Merrill E. , Cohen R. , Salzman E.
Published: May 2019

The reactivity of potentially thromboresistant polymers was determined in sheep by measurement of the lifespan of Cr51-labelled platelets. Materials were tested as tubing (90-125 cm × 2.5-3 mm I.D.) interposed in a 7 cm silicone rubber carotid A.-ext. jugular V. shunt.

Platelet lifespan decreased as the length of the basic silicone rubber shunt increased from 7 cm (“shunt control”, T½ 78.3 ± 11.6 S.D. hrs.; n = 18) to 50 cm (T½ 62.0 ± 7.5 hrs.; n=7; p< 0.001 vs. shunt control) but was longer than in sheep without a shunt (T½ 63.1 ± 11.2 hrs.; n=21; p<0.001 vs. shunt control), probably because of artifactual activation of platelets in blood sampling by needle puncture in the absence of a shunt. Of sixteen polymers studied in 42 sheep (n=117), all significantly shortened platelet lifespan except polyurethane (T½ 72.8 ± 12.1 hrs.; n=12) and non-cross-linked silica-free polydimethyl siloxane (T½ 69.8 ± 13.9 hrs.; n = 9). Polymethyl acrylate (PMA) (T½ 71.2 ± 5.4 hrs.; n=ll; p<0.04 vs. shunt control) was passivated by exposure to platelet free plasma before whole blood (T½ 90.3 ± 3.6 hrs.; n=5; ρ <0.05 vs. untreated PMA). Thus, the adsorbed film of plasma constituents has a lasting and decisive effect on thromboresistance of the surface. The long term behavior of an artificial surface may be dictated by the events of the first few seconds of blood-surface contact.