bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Fibrinogen Binds Edta And Citrate
Authors: Nieuwenhuizen W , Vermond A , Hermans J
Published: July 2018

It was shown that EDTA and citrate make the carboxy-terminal parts of the γ-chains of fibrinogen more susceptible to plasmin degradation. This is an effect independent of the calcium-chelating properties of those compounds. Furthermore, EDTA prolongs the thrombin times decreases the heat stability and causes the formation of abnormal clots.

This suggests a direct interaction of EDTA (and possibly also of citrate) with the fibrinogen molecule thereby causing a conformational change. The interaction of citrate and EDTA with fibrinogen was measured by direct binding studies (equilibrium dialysis).

It was found that at pH 7.5 human fibrinogen binds 3.4 molecules of citrate with Kd = 1.4 x 10-4M or 0.4 molecules of EDTA with Kd = 2.2 x 10-5M. Citrate and EDTA compete for the same binding site(s). No binding of acetate could be demonstrated.

The binding of EDTA and citrate shows a strong ^-dependence suggesting a (partly) ionogenic binding between charged areas on the fibrinogen molecule and charged groups on EDTA or citrate

Our results support the idea that the binding of EDTA and citrate and the concomitant effect on the plasmin attack are independent of the effect of calcium ions
Regulation Of Fibrin Formation
Authors: Blombäck B , Kudryk B , Okada M , Söderman S , Therkildsen L
Published: July 2018

Nossel et al. have shown that fibrin formation in vivo may be regulated by release of the Bβ 1-42 fragment from fibrinogen by plasmin. We have studied the effect of trypsin on the fibrin formation in vitro. Fibrinogen solutions (1 g/1) in Tris-imidazole buffer, pH 7.4, were incubated with trypsin (1:2,000, w/w) for 1-10 min. After incubation soybean trypsin inhibitor was added and clottable fibrinogen recovered by adding thrombin. Non-clottable fibrinogen in the supernatant was recovered by adding equal volume of ethanol. SDS-PAGE, before and after reduction, was performed on the above protein fractions as well as N-terminal analysis on N-DSK obtained from them by treatment with CNBr. Clot-supernatants were used in radioimmunoassay (RIA) for estimation of the following fragments: FPA, FPB, Bβ 15-42 and Hi2-DSK (peptide from COOH-terminal part of α achain). It was found that during incubation the fibrinogen progressively lost its coagulability. The clottable fraction appeared to have the same SDS-PAGE patterns as for intact fibrinogen. The non-clottable fraction showed progressive diminution of intact A α-chains but no degradation products could be observed. The B γ-chain on SDS-PAGE appeared to be shifted towards the Y-chain. The RIA assays showed that concomitant with loss of coagulability there was release of peptides containing Bβ 15-42. Release of FPA and FPB occurred during tryptic digestion but no clotting occurred. Only small release of Hi2-DSK immunoreactivity occurred. NH2-terminal analysis of N-DSK suggested that Bβ 42-43, Bβ 54-55 and a third, as yet unidentified bond, had been split by trypsin. These results suggest that tryptic cleavages in the Bβ and Aα chains destroy the polymerization sites which are unfolded in the molecule by release of FPA and FPB. Similar studies employing plasmin are now in progress.
Prenatal Exclusion Of Haemophilia B By Assay Of Fetal Factor IXC and IXAg
Authors: Mibashan R S , Rodeck C H , Holmberg L , Thumpston J K , White J M , Gorer R , Nilsson I M , Campbell S
Published: July 2018

Recognition of haemophilia B in utero has lagged behind that of classical haemophilia because of lower prevalence and wide emphasis on technical constraints. Pure fetal blood has been obtained at 18-20 weeks by fetoscopy (C.H.R) from 49 of 50 consecutive male fetuses at risk of haemophilia; 4 were pregnancies in obligate or strongly putative carriers of severe haemophilia B.

Current immunoradiometric assays for VIII:CAg depend on both the availability of high titre human antibodies directed against VIII:C and the possibility of preparing highly specific 125I-IgG from human antiserum

Plasma factor IXC in 40 control fetuses was 5.9-12.8 (mean 8.4) U/dl. A sensitive immunoradiometric assay (IRMA) for factor IXAg in 20 of these fetal plasmas gave values of 2.9-7.8 (mean 4.1) U/dl, and 1.8-10.0 U/dl in 9 other control fetuses.

The 4 fetuses at risk of haemophilia B had plasma IXC levels of 7.9, 12.3, 9.4 ö 6.6 U/dl; they were regarded as unaffected, and the parents were counselled accordingly. To date, 3 of the 4 have been born and their clinical and laboratory normality confirmed. The corresponding IXAg levels (Malmö) were also normal — 3.6, 5.4, 4.8 ö 2.0 U/dl; but, despite absence of extraneous IXAg in these samples, exclusion of haemophilia B was not feasible in 2 of the 4 whose affected relatives were found to be CRM

Using pure cord blood obtained at fetoscopy, prenatal diagnosis by coagulant bioassay promises to be definitive and is not limited to CRM fetuses. The IRMA is valuable for diagnosis or confirmation in CRM cases, and in the rare instance (2%) of dilution by amniotic fluid.
The Use Of Non-Invasive Testing And Venography For The Diagnosis Of Acute Recurrent Deep Vein Thrombosis
Authors: Hull R , Carter C , Ockelford P , Hirsh J , Zielinsky A , Turpie A G G , Powers P
Published: July 2018

Impedance plethysmography (IPG) combined with leg scanning is highly sensitive and specific for deep vein thrombosis (DVT) in patients with their first episode of clinically suspected DVT. This approach has not been evaluated in patients with suspected recurrent DVT. In this group of patients venography alone is of limited value in excluding acute DVT in the presence of previous DVT but is useful if constant intraluminal filling defects (ILFD) suggesting acute DVT are present. We have evaluated the clinical utility of IPG plus leg scanning and venography in 132 patients with clinically suspected acute recurrent DVT. If the IPG on referral was negative the patient was leg scanned daily for 72 hours and if both were negative, anticoagulant therapy was witheld. The validity and safety of this approach was tested by 3 months follow-up. If IPG was positive, venography was performed to distinguish ILFD’s from chronic DVT. If ILFD’s were detected, anticoagulant therapy was commenced. If no ILFD’s were detected the patient was leg scanned for 72 hours and if negative treatment was witheld. The clinical utility of this non-invasive approach is demonstrated by the results of this study. Of 132 patients, 82 (62%) were negative by both non-invasive tests and none died, developed pulmonary embolism or recurrent DVT during follow-up. The remaining 50 patients were positive by noninvasive testing; in 31 patients ILFD’s were detected and in 16 venography was indeterminate showing collaterals, absent segments, recanalization or inadequate visualization. The leg scan was positive in 10 of the latter 16 patients. Thus the diagnosis of recurrent acute DVT was established by the presence of ILFD’s or a positive leg scan. In 6 patients with a positive IPG, negative leg scan and indeterminate venogram the diagnosis remained uncertain. In conclusion this combined non-invasive and invasive approach provided definitive management in 126 of 132 patients (95%).
Noninvasiv Screening For Acute Dvt By Means Of Leg Temperature Profiles And Venous Emptying Curves As Compared To Conventional Thermography And Phlebography
Authors: Nilsson E , Holmgren K , Jacobsson H , Johnsson H , Zetterquist S
Published: July 2018

simplified thermographic technique has been developed to provide leg temperature profiles for diagnosis of acute DVT (DevethermR). The temperature profiles are obtained by manual scanning from the foot to the hip with an infrared radiation transducer attached to a position indicator. The diagnosis of DVT is based on side-differences in mean segmental leg temperature.

In 200 patients admitted to hospital due to the clinical suspicion of acute DVT, the leg temperature profiles were pathological in 132 of those 140 patients who proved to have DVT at phlebography which means a sensitivity of 94%. False positive results were obtained in cases with e.g. varicose veins or regional infections but these errors could be reduced if the profiles were not only numerically evaluated but also subjected to pattern analyses. A combination with venous emptying curves as obtained from venous occlusion plethysmography further improved the specificity of the noninvasive diagnostic procedure. This combination also proved favourable for the differentiation between proximal and distal leg thromboses as well as acute and old DVT. Some of the patients were investigated with thermography by a scanning camera. The results correlated closely to those obtained with the simplified thermographic technique but without improving the diagnostic sensitivity.
Success Rate Of Fibrinolytic Therapy In Fresh And Old Deep Vein Thrombosis
Authors: Theiss W , Wirtzfeld A , Maubach P
Published: July 2018

It is still a wide-held belief that fibrinolytic therapy can clear thrombi from deep veins only while they are fresh. We have therefore analysed retrospectively the phlebographic results obtained in 85 patients, all of whom had fibrinolytic therapy for thrombosis of the iliac and/or femoral veins with symptoms present for 1 day to 8 weeks prior to treatment. Streptokinase (n=46), urokinase (n=9), or both drugs successively (n=30) were administered according to our guidelines as recently outlined in detail (Klin. Wschr. 58:521, 1980; Med. Klin. 75:580, 1980) particular emphasis being placed on sufficient duration of thrombolytic therapy with no a priori time limit.

Thus, S.C. administration of heparin seems to be as efficient and safe as administration of heparin I.V. in the treatment of patients with acute DVT.

The number of cases grouped according to the lenghth of their history and their respective phlebographic outcome as well as the duration of thrombolytic therapy are listed in the table.

It can be seen that the success rate was good, when the patient presented with a delay of up to 2 weeks; even during the third or fourth week of the thrombotic episode two thirds of the patients could still be improved. Thereafter the results were uniformly poor.

Conclusion: Iliofemoral venous thrombosis can be treated successfully with fibrinolytic drugs for much longer after its onset than is generally accepted.
Effects Of Exogenous Calcium On Ionophore A23187- Induced Platelet Stimulation
Authors: Lages B , Kruger C , Weiss H J
Published: July 2018

The ionophore A23187 is known to induce platelet (P) secretion in the presence or absence of extracellular calcium(Ca), thus providing evidence that endogenous Ca participates in the secretory mechanism. To explore further the interaction of A23187 with P Ca, we have compared the P responses to A23187 in the presence and absence of extracellular Ca using stirred gel filtered P in Ca-, Mg-free Tyrode’s containing EGTA or EGTA plus Ca. With Egta alone, A23187 induced an immediate increase in light transmission(LT) which was not associated with aggregation, and a rapid secretioiji of 14C-5HT, ATP+ADP, and acid hydrolases which was complete within 30 sec. Increasing Ca, however, caused a progressively greater lag in secretion response(up to 60 sec.) but did not decrease the extent of secretion. With Ca/EGTA, LT changes were associated with visible P aggregation. In vivo ASA treatment reduced the extent of both secretion and aggregation but did not alter the effect of Ca on the secretion time course. Similar patterns of LT changes with EGTA vs aggre-i- gation with Ca/EGTA occurred in response to thromi- bin, but Ca did not cause comparable effects on the time course of thrombin-induced secretion. These results suggest that the P response to A23187 when intracellular Ca is utilized differs from that when Ca is supplied externally and that this is not due solely to the presence of aggregation. We hypothesize that these different responses may reflect changes in an endogenous P Ca pool which participates in P secretion.
Calmodulin Binding Proteins In Platelet Actomypsin
Authors: Muszbek L , Harsfalvi J
Published: July 2018

Platelet actomyosin (thrombosthenin) possesses a myosin-linked Ca2+ regulation and Ca2+ sensitivity is conferred to it by calmodulin through myosin light chain kinase. Calmodulin binding proteins if they are present in the actomyosin complex may have an important regulatory role in the contractile mechanism of platelet activation. To test this possibility an aceton powder was made from platelet actomyosin and extracted with an 8 M urea containing buffer. The extract was examined for the presence of calmodulin binding proteins by alkaline urea polyacrylamide gel electrophoresis. It was shown by this technique that some proteins in the actomyosin complex can form a Ca2+ dependent complex with both calmodulin and skeletal muscle troponin C (TNC is closely related to calmodulin) even in the presence of 8 M urea. Calmodulin binding proteins could be isolated from the extract by affinity chromatography in 8 M urea on TNC-Agarose column. 3 major proteins of 270 K, 6l K and 23 K molecular weight were eluted by EGTA and each of them was able to bind to calmodulin or TNC if Ca2+was present. At least one of these calmodulin binding proteins exerted a troponin I like effect when tested on reconstituted skeletal muscle actomyosin and the 23 K protein showed a close similarity to troponin I. the inhibitory protein of the actin linked Ca2+ regulatory system in skeletal muscle. It is presumed that calmodulin binding proteins may have a dual role in the regulation of platelet actomyosin. They can inhibit the Ca2+ dependent phosphorylation of myosin light chain and one or more of them may also exert an actin linked inhibitory effect.
Hemorheological Effect Of Ticlopidine In Rats
Authors: Ono S , Ashida S , Abiko Y
Published: July 2018

The hemorheological effect of ticlopidine was studied in rats ex vivo. Ticlopidine (30-300 mg/kg) was orally given to rats. Heparinized blood samples were taken from the carotid artery under pentobarbital anesthesia 3 hr after the drug administration for measurement of whole blood viscosity (ELD type cone-plate viscometer), micropore filtrability of red cells (Nuclepore membrane, 5 µm), erythrocyte sedimentation rate (ESR), hematocrit (Ht) and plasma fibrinogen. Red cell deformability was measured by counting the shear stressinduced cap-form cells under a scanning electronmicroscope. Mechanical flexibility of red cells was also studied by measuring hemolysis caused by turbulant flow.

Ticlopidine treatment caused a significant decrease in whole blood viscosity (9.13 ± 0.15 and 6.17 ± 0.08 versus 9.80 ± 0.18 and 6.74 ± 0.09 Cp in control at 19.2 sec-1and 76.8 sec-1, respectively) and a significant increase in micropore filtrability of the red cells (0.54 ± 0.01 versus 0.40 ± 0.02 ml/min in control) without any changes in ESR, Ht and plasma fibrinogen. Ticlopidine also significantly stimulated the shear stress-induced shape change of the red cells to cap-form cells (12.08 ± 0.13 versus 8.66 ± 0.23 % in control) and prevented mechanical hemolysis caused by a turbulant flow (16.8 ± 1 . 6 versus 30.5 ± 2.5 % in control).

In addition to the platelet aggregation inhibitory action the hemorheological action of this agent may be useful for improving microcirculation and protecting red cells from mechanical disruption by turbulant blood flow.

Increase in the adenylate cyclase and Mg2+-activated adenosine triphosphatase activities in red cell membranes may be associated with the effect of ticlopidine to increase red cell deformability.
Thrombin Noncovalent-Protein Binding And Fibrin(Ogen) Recognition
Authors: Fenton J W , Zabinski M P , Hsieh K , Wilner G D
Published: July 2018

Procoagulant α-thrombin is a highly selective proteolytic enzyme with central bioregulatory functions in hemostasis. Unlike with plasma protease inhibitors, human α- thrombin forms a noncovalent, yet high-affinity complex with hirudin (Ki∼ 6.3 pM), whereas noncoagulant γ-thrombin has ∼200-fold reduced affinity. Like 125I-α-thrombin alone, 125I-DIP- or 125I_pMS-α-thrombin in the presence of unlabeled α-thrombin incorporates into fibrin clots, but 125I-γ-thrombin or the hirudin complex of 125I-α-thrombin does not. Similarly, α-thrombin and its catalytic-site conjugated forms (e.g., DIP-α-thrombin) bind to nonpolymerized fibrin agarose and elute at higher ionic strengths (I ∼0.2 M) than do noncoagulant nitro-α- or γ-thrombin. Hirudin prevents this binding of α-, DIP-α-, or γ-thrombin, demonstrating a common site. The NDSK fragment of fibrinogen is a moderate inhibitor of the esterolytic activity of a-thrombin (Ki∼ 1 µM), while the residue-17 through -27 synthetic homologue of the fibrinogen Aa chain is not an inhibitor. These results suggest contiguous exosites in thrombin for noncovalent-protein binding and fibrin(ogen) recognition, where these sites are removed from the enzyme catalytic site and correspond to structural regions on the fibrin side of fibrinogen other than those of the α-chain NH2- terminus required for fibrin polymerization. Such binding/- recognition sites may explain the physiological proteolytic and receptor specificity of α-thrombin.
The Prothrombin Time: The Mechanism Of In Vitro Activation
Authors: Palmer R , Gralnick H
Published: July 2018

We have described shortening of the prothrombin time (PT) when blood is collected in borosilicate or siliconized boro- silicate tubes. The shortening is time dependent and occurs more rapidly at 4° C than at room temperature. We have studied the mechanisms of in vitro activation of the PT utilizing normal blood and blood congenitally deficient in coagulation factors. We have found that the blood deficient in factor XI has the same rate of shortening of the PT as in normal blood, while blood deficient in factor IX has only 50/ that of normal and factor XII deficient blood does not have any in vitro activation. The blood deficient in Cl INH ihas the most severe shortening of the PT.

Analysis of the coagulation factors affected during the in vitro activation of normal blood revealed that factor VII-X is increased and this is related primarily to factor VII activation. In borosilicate factor V did not change, factor IX decreased by 15-20% while factor XII showed no change in the assay system. The activation can be totally blocked by addition of Cl INH or corn trypsin inhibitor (specific inhibitor of XII activation) to whole blood. These studies indicate that the PT is shortened in vitro by at least two mechanisms of activation of whole blood: 1) factor XII is activated by surface contact with siliconized borosilicate or borosilicate and in turn, directly activates factor VII; 2) factor XIIa activates prekallikrein to kalli- krein which in turn activates factor IX. Factor IXa then activates factor VII and it would appear that approximately one-half of the activation of the PT is directly through this pathway. Inhibitors of factor XII activation, Cl INH and CFI can totally inhibit the activation in vitro. Thus, surface activation of factor XII is the pivotal reaction in the in vitro shortening of the PT in whole blood. These studies allow new strategies for the prevention of in vitro activation of the PT, and may allow for more precise measurement of the PT without concern for contact activation by the use of specific inhibitors.
Influence Of Cod Liver Oil And Corn Oil On Platelets And Vessel Wall In Man
Authors: Nordøy A , Brox J H , Killie J E
Published: July 2018

Two groups of 10 healthy males were given dietary daily supplement of 25 ml cod liver oil (CLO) or corn oil (CO) for periods of 6 weeks in a blind crossover study. Between the two periods and after the last period there were 3 weeks and 4 months, respectively, without supplement. Plasma lipids, platelet lipids, prostaglandins and platelet functions were examined. Pieces of superficial forearm veins were excised after the two dietary periods and assayed for prostacyclins by bioassay and by GLC-MS.

Collagen-induced platelet aggregation and TXB2 formation were significantly reduced in both the CLO and CO group. CLO was more potent than CO. The MDA production was not affected.

In the total fatty acids of plasma and in the main platelet phospholipid fractions, CLO increased the amounts of fatty acids of the w-3 familiy whereas the w-6 fatty acids decreased. CO only moderately influenced the composition of these lipids. Cholesterol and triglycerides levels in serum were not affected. The vein biopsies showed no significant production of prostacyclin-like material in any of the dietary groups. In summary small amounts of CLO added to the diet gives significant changes in platelet lipids and reduced platelet aggregation and TXB2 production.
Fibrinopeptide A Cleavage And Platelet Release In Whole Blood In Vitro
Authors: Kaplan K L
Published: July 2018

The relationship between platelet release and fibrinopeptide A (FPA) cleavage from fibrinogen to form fibrin I was examined in blood allowed to clot in vitro. FPA and the platelet release products (platelet factor 4 and (β-thrombo- globulin) were quantitated by radioimmunoassay. In undisturbed blood platelet release and FPA cleavage occurred simultaneously. Addition of hirudin prevented platelet release as well as FPA cleavage, indicating that thrombin was essential for platelet release. Addition of collagen increased platelet release and then FPA cleavage, and addition of ADP had a similar but less marked effect, suggesting that increased platelet activation accelerated thrombin formation. PGE1 and theophylline inhibited platelet release but did not affect FPA cleavage in the initial stages of incubation. When blood was agitated, the effects of collagen and PGE1 and theophylline were qualitatively similar to those in undisturbed blood, but addition of hirudin no longer affected platelet release, suggesting that in this case platelet release occurred independently of thrombin action. Addition of dilute thrombin or tissue thromboplastin to undisturbed blood accelerated FPA cleavage with little effect on platelet release, indicating that exogenous thrombin or thrombin generated by the extrinsic system behaves differently from that generated by the intrinsic system. Finally, thrombin concentrations were compared in platelet rich and platelet free plasma and were found to be increased in platelet rich plasma in spontaneously clotting blood but not in blood to which tissue thromboplastin was added. These relationships suggest that thrombin formation via the intrinsic system occurs on the platelet surface leading to simultaneous platelet release and FPA cleavage, while thrombin generated via tissue thromboplastin leads to thrombin formation in the plasma and FPA cleavage precedes platelet release. Collagen primarily accelerates platelet release and secondarily increases thrombin formation.
Kinetics Of Anticoagulant And Lipolytic Activity And Radiolabel After Intravenous Administration Of 35S-Heparin Fractions Of Different Molecular Weight
Authors: de Swart C A M , Sixma J J , Nijmeijer A , Holmer E , Andersson L O , Verschoor L
Published: July 2018

Heparin fractions were prepared from commercial pig mucosal by affinity chromatography on antithrombin III, 35S- radiolabelling and gelchromatography. Three fractions were obtained with moLwt.of < 6.000, 6 - 35.000 and > 35.000 daltons. They were administered intravenous as a bolus into human volunteers. The anticoagulant activity was measured with the APTT and Xa inactivation, using standard curves. The lipolytic activity was as^ed as hepatic triglyceride lipase activity, and as extra-hepatic lipoprotein lipase activity by hydrolysis of tritiated triolein. Radioactivity data were corrected for degraded heparin fragments uncomplexed to protein by subtracting radiolabel with a mol.wt. < 10.000 passing through a filter.

Low mol.wt. fractions induced neither of both lipase activity and had no effect on the APTT. Anti Xa activity and radioactivity disappeared in parallel with slightly concave curves in semilogarthmic plots. Heparin of intermediate and high mol.wt. induced both lipolytic and both anticoagulant activities. The elimination of radioactivity, hepatic triglyceride lipase activity and anti-Xa activity occurred parallel according to a convex curve in semilogarthmic plots. The extra-hepatic lipoprotein lipase activity disappeared following a slightly concave curve.

These data indicate that relatively large heparin molecules are required for lipolytic and APTT activity. Hepatic triglyceride lipase activity might be present complexed to heparin, comparable to the antithrombin III-heparin complex. The elimination of anti Xa activity and hepatic triglyceride lipase activity might be determined by the heparin part of the complexes.
Antithrombotic Properties Of Low Molecular Weight Heparin Fractions From Porcine Mucosal Heparin
Authors: Choay J , Lormeau Jean C , Messmore Harry L , Fareed Jawed , Stulc J , Andersen Anne
Published: July 2018

A previous report from our laboratories has described the extraction and physicochemical properties of a low molecular weight fraction (mol wt 4-8 × 103 daltons) from porcine mucosal heparin (Choay et. al. thrombosis Res 18, 573, 1980). Beside exhibiting a strong anti Xa (>250 u/mg) activity, this product possessed strong antithrombotic properties in a modified rabbit stasis thrombosis model. At a 125 anti Xa u/kg it protected the thrombotic effects of activated prothrombin complex concentrate (20 u/kg) and Prothrombin Complex Concentrate/Russell’s Viper Venom challenge in both the pretreatment and post-treatment regiments. At 1250 anti Xa u/kg SC it also showed antithrombotic effects for various periods. We have also obtained another low molecular weight fraction from porcine mucosal heparin by controlled depolymerization with nitrous acid. This product possessed saccharides with molecular weight ranging 3-6 × 103 daltons and exhibited a specific activity of >200 anti Xa u/mg. At a 125 anti Xa u/kg this product also showed antithrombotic activity against the thrombotic effects of activated prothrombin complex concentrates, prothrombin complex concentrates and Russell’s Viper Venom. In contrast to these two low molecular weight fractions porcine mucosal heparin in identical anti Xa units failed to produce protection against the thrombogenic stimuli. Our studies suggest that low molecular weight heparin fractions with strong anti Xa and antithrombotic activities can be obtained by chemical depolymerization. Furthermore, their biologic properties are found to be similar to the naturally occuring low molecular weight fractions present in native porcine mucosal heparin.
Anti-Platelet Therapy In Diabetic Patients With Retinopathy
Authors: Tindall H , Paton R C , McNicol G P
Published: July 2018

The effect of a combination of aspirin at 2 dosage levels (330mg/day and 1gm/day) and dipyridamole 225mg/day on platelet lifespan and some haemostatic variables was investigated in 30 insulin-dependent diabetic patients with retinopathy. Platelet regeneration time using a modification of the Stuart technique was measured in 40 normal controls and all patients before therapy commenced. Treatment was then allocated in a double-blind manner using a Latin square design and haemostatic variables were measured at the end of each treatment period. Platelet survival by the standard Na51Cr radioisotopic technique was performed twice during treatment. The post-aspirin platelet regeneration time in normal subjects was 9.98 ± 0.22 days (mean ± SEM) and in diabetic patients was significantly (p<0.001) shorter at 7.16 ± 0.18 days. A similar result of 7.37 ± 0.20 days was obtained in the diabetic patients by Na51cr platelet survival (analysed by γ function). The aspirin/dipyridamole combination (330mg/75mg tds) resulted in significant (p<0.001) lengthening of platelet survival to 8.46 ± 0.13 days (as estimated by Na51Cr method). The postaspirin platelet regeneration time was significantly prolonged (p<0.01) in this group (9.75 ± 0.52 days).

Although the lower dose aspirin - dipyridamole combination (110mg/75mg tds) caused a prolongation in regeneration time (8.29 ± 0.45 days) this time was not significantly different from placebo, and was significantly (p<0.01) shorter than the time taken on the higher aspirin dosage. No significant changes in haemostatic function were observed. These results support the hypothesis that platelets may be involved in the microvascular complications of diabetes mellitus. Treatment with Igm aspirin +225mg dipyridamole/day significantly lengthened platelet survival suggesting that long term trials at this aspirin dosage level are justified.
Investigation Of The Origin Of The Density Heterogeneity Of Rabbit Platelets
Authors: Rand M L , Packham M A , Mustard J F
Published: July 2018

Platelets are heterogeneous with respect to size, density and function. The question is unsettled whether platelets of different size and density are produced from megakaryocytes or whether large, heavy platelets become less dense as they circulate and are exposed to aggregating agents that decrease platelet density such as thrombin, plasmin, or ADP. Platelet subpopulations were separated by discontinuous Stractan density gradient centrifugation. Most dense platelets were larger than least dense platelets (n=5, p<0.0025). After labeling platelets in vivo with 35SO4
2-, the incorporation and loss of 35S in platelet density subpopulations was determined (n=5). At 1 hr, no 35S was detected in the platelets. By 24 hr, 35S was present in all density subpopulations but the relative specific radioactivity (RSR) of the most dense subpopulation was 7 times greater than that of the least dense, although the most dense platelets were only 1.2 times larger. The RSR of the most dense subpopulation reached a maximum between 48 and 72 hr and then decreased gradually whereas that of the least dense increased slowly, reaching its maximum at 96–120 hr. These results support the theory that platelets become less dense as they age. 51Cr-labeled platelets were injected into rabbits and the loss of radioactivity in platelet density subpopulations was determined (n=3). The most dense subpopulation decreased in RSR more quickly than the least dense. The % radioactivity/% platelets of the least dense subpopulation increased from 0.58 to 0.82 while that of the most dense decreased from 1.46 to 1.13 over 72 hr. These observations could be due to either preferential removal of heavy platelets or decrease in density on aging. Although these results support the hypothesis that the majority of platelets released into the circulation are larger, more dense platelets which decrease in density on aging, platelets of all densities are released into the circulation. Thus the origin of platelet density heterogeneity cannot be ascribed solely to aging, although this does contribute to it.
Human Platelet Age Correlates With Buoyant Density, But Not With Size
Authors: Mezzano D , Catalano P , Hwang K , Aster R H
Published: July 2018

Following infusion of 51Cr-labelled autologous platelets into normal subjects, high density (HD) and low density (LD) platelet cohorts were isolated by centrifugation in isosmotic arabino- galactan (Stractan) . Specific radioactivity (SA) of LD platelets declined rapidly post-infusion (T1/2 =1.8 days) but SA of HD platelets remained constant or increased over a 3-4 day period and gradually declined for 6-7 days thereafter. These differences were exaggerated when platelet cohorts enriched in LD or HD cells by slow centrifugation in high density albumin were labelled and transfused. Mean survival of a platelet cohort enriched with HD cells was significantly (p < .02) shorter (7.73 days) than that of a cohort enriched with LD cells (9.33 days). In normal subjects treated with aspirin, capacity for thromboxane synthesis was regained more rapidly (p < .05) in LD than in HD platelets. HD and LD platelets differed only slightly in mean volume (HD platelets = 7.57 μ3, LD platelets = 6.87 μ3, 0.05 < p < 0.01).

These findings imply that under normal conditions in man, newly formed platelets are less dense on the average than total platelets and become more dense as they age in the circulation. Thus, SA of LD platelets declines rapidly as these platelets move into a more dense compartment and are replaced by newly formed, unlabelled platelets; SA of HD platelets remains constant or increases as labelled platelets enter this compartment in numbers equal to or greater than the number leaving it at the end of their life span. The similarity in mean volumes of LD and HD platelets suggests that platelet size is unrelated to platelet age under normal conditions
Effect Of Age And Operations On Platelet Serotontin (5Ht) And Platelet Volume
Authors: Shuttleworth R D , O’Brien J R
Published: July 2018

To pursue the concept that “activated” platelets (plats) in vivo might be deficient in 5HT (as well as being deficient in PF4 - Abstract submitted), we collected citrated blood from 39 “healthy” controls aged 15–50 yrs and from 14 patients (25–75 yrs), 2–8 days after major surgery. The plat 5HT, the 5HT uptake and plasma 5-hydroxyindoles (PPP 5HTs) were measured fluorometrically and the mean plat, volume (MFV) was measured using a Coulter S plus counter.

In health (n = 39) with increasing age the MPV (correlation: r = 0.45, p<0.01), the plat 5HT (r = 0.62, p<0.001) and 5HT uptake (r = 0.45, p<0.0l) all decreased while the PPP 5HIs increased (r = 0.71, p<,0.00l). The plat 5HT was also positively correlated with 5HT uptake (r = 0.42, p<0.01) and with MPV (r = 0.45, p<0.01) and negatively with PPP 5HIs (r = 0.43, p<0.01).

After operations (n = 14) irrespective of age the plat 5HT was lower than the controls' (p<.0.001) and the MPV and 5HT uptake were low, but not significantly so. The plat, count was significantly lower than the controls (p<0.001). Again the plat 5HT correlated with MPV and 5ê uptake.

Differential centrifugation of normal PRP into platelet sub-populations with differing MPVs confirmed the correlation between MPV and plat 5HT.

It is concluded that both operations and increasing age are associated with the appearance of small relatively empty plats “exhausted” of 5HT which cannot take up 5HT. Have these plats been partially activated and undergone release with a subsequent decrease in volume? Alternatively, are large platelets relatively full of 5HT removed from the circulation, or not produced? (These findings support and extend the findings of Shuttleworth et al, Blood March 1981: in press).
Factor VIII In Vessel Wall Influences Platelet Interaction With Subendothelium
Authors: Turitto V , Weiss H , Sussman I , Zimmerman T
Published: July 2018

Immunological techniques have demonstrated the presence of factor VIII:AGN in both the endothelium and deeper layers of vessel wall. Since plasma factor VIII:VWF is known to mediate platelet adhesion to subendothelium, we investigated the influence of vessel wall factor VIII on platelet interaction with the vascular surface. Everted vessel segments (14 mm in length) from rabbit aorta, denuded of their endothelial lining by a balloon catheter technique, were incubated for 1 hr at 37°C in 1ml of either 0.2M Tris buffer (B), normal goat serum diluted 1/5 with B or goat serum containing an antibody to rabbit factor VIII:VWF diluted 1/5, 1/20 or 1/100 with B. The segments were exposed in an annular chamber to citrated huipn blood, flowing at 40ml/min (wall shear rate of 2600 sec-1 ) for 5 min. The percentage of subendothelial surface covered with adherent platelets (A) or platelet thrombi greater than 5μm in height (T) were determined morphometrically. Values of A were significantly decreased on vessels treated with serum containing antibody diluted 1/5 (A=30.4±5.9, p<0.0l) or 1/20 (A=31.7±5.0, p<0.0l) but not 1/100 (A=37.7±7.4, p=NS) compared to vessels incubated with either B(A=53.8±5.l) or normal serum (A=58.0±7.9). Thrombus formation (T) was not significantly decreased by treatment of the vessel wall with serum containing antibody. Similar results were obtained when the IgG fractions of serum were used instead of whole serum.

Thus, it appears that factor VIII:AGN in the vessel wall may be important in the initial platelet events involved in hemostasis. The relative importance of vessel wall factor VIII versus that present in the plasma requires further study.
Effect Of An Arginine-Modifying Reagent On The Responses Of Rabbit Platelets To Agonists
Authors: Anderson E R , Foulks J G
Published: July 2018

Arginyl groups participate at the active binding site of several enzymes with anionic substrates, particularly adenine nucleotides. Hence, such groups are reasonable candidates for the cationic groups at membrane surfaces which contribute to the binding of the phosphate groups of ADP to platelet receptors. To investigate the importance of surface arginyl residues to the aggregation responses of platelets, an impermeant analogue of the arginine-modifying reagent phenylglyoxal, p-sulfonyl phenylglyoxal (PSPG), was synthesized. Rabbit platelets were exposed to this reagent during an extra washing step inserted in the usual resuspension procedure. Pretreatment of resuspended rabbit platelets with 2-15 mM PSPG resulted in total inhibition of aggregation responses to ADP, and to 5-HT, and a concentration-dependent impairment of the preceding shape change responses. Aggregation responses to thrombin (which preferentially cleaves peptide bonds involving arginine and lysine) were inhibited in a concentration dependent manner. To determine whether the inhibition of ADP-induced aggregation by PSPG is non-specific or is a selective effect due to modification of arginyl groups at the active binding site for ADP, resuspended platelets were pretreated with inactive analogues of ADP during treatment with PSPG. The presence of either ATP or β,γ-methylene ATP during pretreatment with PSPG provided significant or complete protection against the inhibitory effects of PSPG on ADP responses, and also partial protection of the responses of platelets to thrombin. Neither of these agents was effective in protecting the ability of platelets to respond to 5-HT.

These experiments indicate that intact arginine residues at the surface of rabbit platelets are necessary for their responsiveness to ADP, thrombin and 5-HT, and that these residues form part of the ADP receptor.
Relation Of Heparin Binding And Conformation In PF4 AND LA-PF4 (βTG)
Authors: Holt John C , Kloczewiak Marek , Walz Daniel A , Rucinski Boguslaw , Niewiarowski Stefan
Published: July 2018

Platelet factor 4 (PF4) and low affinity platelet factor 4 (LA-PF4) are platelet-specific secreted proteins that bind to heparin. β-thromboglobulin (βTG) appears to be derived from LA-PF4 by proteolysis of four NH2-terminal residues. PF4 and LA-PF4 (βTG) show 50% sequence homology including four cysteine residues and two pairs of lysine residues near the C00H-terminus which are believed to be responsible for heparin binding. Despite these similarities, the two proteins have markedly different affinities for heparin. We have sought a structural interpretation of this difference by predicting the conformations of 0TG, LA-PF4 and PF4. First, the proportion of residues in α-helical, β-sheet and unordered conformations was estimated from circular dichroism measurements. The results for PF4 and LA-PF4 were experimentally identical, namely 16% α-helix and 20% β-sheet. These values were then applied as experimental constraints in the prediction of the secondary structure of PF4 and LA-PF4 based on their amino acid sequences. This was done by a computer program which compared local amino acid sequence (each residue and 8 residues on either side) with the conformation of similar sequences in 25 proteins of known structure. With the further constraint of the two disulfide bonds in each molecule, models were constructed representing the overall folding of the polypeptide chains. The only significant difference between the two proteins was in the COOH-terminal region of the chains. The models suggest that the lower affinity of LA-PF4 (and βTG) for heparin may result from steric hindrance by the longer and more negatively charged COOH-terminal segments of these molecules compared with PF4.
Human Platelet Aggregation In Response To Multiple Agonists In Plasma Anticoagulated With Heparin
Authors: Culliver H A , Ardlie N G
Published: July 2018

The lowest concentrations at which epinephrine and vasopressin have been reported to interact positively in causing platelet aggregation in vitro are at least two orders of magnitude greater than the physiological concentrations of these hormones in blood. The aim of this study was to examine the interaction between several agonists of human platelet aggregation. The aggregating agents used were adenosine diphosphate (ADP), epinephrine, norepinephrine, 5-hydroxytryptamine and vasopressin. Platelet-rich plasma (PRP) was prepared from blood anticoagulated with minimal concentrations of heparin in an attempt to more closely reflect the in vivo situation.

Aggregation caused by ADP was potentiated by epinephrine at a concentration exceeding the level obtained in circulating blood. When a third agonist (vasopressin) was used in combination with ADP and epinephrine, aggregation was enhanced at concentrations of vasopressin and epinephrine obtained in blood. When used as a fourth agonist norepinephrine and 5-hydroxytryptamine potentiated aggregation at physiological concentrations. The response to multiple agonists was greater in heparinized PRP than citrated PRP. Hirudin decreased the extent of aggregation in heparinized PRP caused by multiple agonists suggesting that thrombin may be involved.

Since the concentrations of combined agonists required to induce in vitro platelet aggregation can be obtained in circulating blood these findings may explain why platelet activation occurs in certain pathological states.
Effect Of Novel PGEl Analogue (OP-1206) On The Platelet Functions
Authors: Kitani T , Nakagawa M , Maeda Y , Kavamura T , Watada M , Yoshikawa H , Hino Y , Ijichi H
Published: July 2018

PGEl is one of prostaglandins which inhibit platelet functions and has vasodilating activity similar to PGI2. Newly developed PGEi analogue (OP-1206) was supplied for the clinical evaluations on oral administration. This research was performed to analyze the effect of orally administrated PGE1 analogue on platelet functions and to evaluate the usefulness on the thromboembolic disorders. Comparing with PGI2, this analogue demonstrated the similar inhibitory activity on the platelet aggregation in vitro study. Oral administration of OP-1206 on the patients with thromboembolic disorders showed the dose-dependent inhibition on platelet aggregation and adhesiveness. This activity continued for l80 min (max at 120 min). Daily oral administration (20μg and 30μg t.i.d.) was continued for two weeks and its effect on blood pressure, heart rate, ADP induced platelet aggregation, platelet adhesiveness and platelet c-AMP level were evaluated. Both of administration doses caused remarkable depression of platelet aggregation, increment of c-AMP level in the platelet and mild suppression on the platelet adhesiveness. Blood pressure was decreased, but heart rate remained unchanged. Clinical improvement of symptoms were observed in the patients with deep vein thrombosis or angina pectoris. These results emphasize the effectiveness and usefulness of this orally administrated PGE1 analogue against the prevention and treatment of thromboembolic disorders.
Dissolution Of Thrombi By Tissue Plasminogen Activator, Urokinase And Streptokinase In An Artificial Circulating System
Authors: Mattsson C , Nyberg-Arrhenius V , Wallén P
Published: July 2018

The ability of porcine tissue plasminogen activator, urokinase and streptokinase to dissolve whole blood clots has been compared in an artificial circulating system, the Chandler loop. On a molar basis the tissue activator was more effective in lysing both partially and totally cross- linked thrombi than urokinase or streptokinase. The latter drugs required 30–50 times higher concentrations to achieve a fibrinolytic response, similar to that of tissue activator. As demonstrated by analysis of fibrinogen, α2-anti- plasmin and plasmin-α2-antiplasmin complex there was no activation of plasminogen in plasma by the tissue activator in the absence of fibrin. Both urokinase and streptokinase gave under the same conditions rise to a significant dose dependent fibrinogenolysis. In the presence of fibrin, tissue activator induced a weak fibrinogenolysis (about 20 per cent) in plasma, whereas both urokinase and streptokinase caused a much higher degree of fibrinogenolysis (50 and 60 per cent respectively)
Physical, Chemical And Immunological Properties Of Porcine And Human Plasminogen Activators Of Various Tissues And Cells
Authors: Cole E R , Snopko R M
Published: July 2018

Properties of plasminogen activator (PA) from heart, kidney, lung and plasma euglobulin of the pig and pig kidney cell culture were compared with the properties of PA from human heart, lung, urinary urokinase (UK) and human kidney cell culture (Abbokinase, AK). The isoelectric points of PA’s were in the pi range of 7.0-7.8, except for UK and AK which had higher pI, 8.8-9.2 for the major forms. Mol. Wt. estimation by gel filtration revealed all PA, except AK, to have Mol. Wt. 48,000-54,000 with additional low Mol. Wt. forms of 25,500 and 36,000 for pig kidney cell culture PA and UK respectively. A single 35,500 form was seen for AK. Antiserum prepared against highly purified pig heart activator quenched the PA activity of pig heart, kidney, lung and euglobulin, but was inactive against pig kidney cell culture PA and UK. While pig heart PA is a poor activator of purified plasminogen and hydrolyzes AGLME poorly, pig kidney cell culture PA, UK and AK rapidly activate plasminogen and have high AGLME hydrolysis activity. A cofactor for pig heart PA (E.R. Cole, 1977, Blood 50: 262) which markedly accelerates plasminogen activation by pig heart PA, does not increase plasminogen activation by pig kidney cell culture PA, UK, AK or pig euglobulin and is instead slightly inhibitory. This data suggest that PA extracted from tissues is primarily in an inactive form, has little plasminogen activation and AGLME hydrolysis activity and is sensitive to cofactor due to unfolding of the PA with exposure of the active site. The urinary and cell culture forms of PA, in contrast, are released from cells in the active form, are insensitive to cofactor and have high plasminogen activation and AGLME hydrolysis activities.
The Activated Partial Thromboplastin Time After Heparin Removal (aPTT/HR): The Basis Of A New Single Test Stratagem For Monitoring All Phases Of Anticoagulation With Heparin And/Or Coumarins
Authors: Branson H E , Engelberg C , Fagin A , Puri S , Broadbrooks J
Published: July 2018

Safe and accurate monitoring of the transition from the antithrombotic protection afforded by the heparin-antithrombin III complex to that of coumarin modified zymogens has traditionally been provided by special dilutional one-stage prothrombin times. The demonstrated ability of ECTEOLA anion exchange columns to remove heparin from plasma containing acarboxy forms of the vitamin k dependent factors led to the development of an alternative test, the protime after heparin removal (PT/HR) (Am J Clin Pathol 71 (6): 665, 1979). Application of this same technology to the activated partial thromboplastin time (aPTT/HR) results in: 1) a superior alternative to dilutional and ion exchange protimes; and 2) a new single test/pathway scheme for following the heparin, heparin and coumarin, and coumarin phases of anti-coagulation therapy. The fitness of the aPTT for monitoring therapy with heparin alone and coumarins alone has been previously established. Preliminary studies indicate that the aPTT/HR is very similar in its ability to determine the adequacy of anticoagulation to a combination of the PT and aPTT. Comparison of the aPTT/HR with the PT/HR for combined heparin and coumarin monitoring indicated that it:
1) more closely approximates the functional activity of factor X; 2) is equally sensitive to heparin and coumarins; 3) is responsive to more common clinically significant depressions of factors; and 4) is the test of choice for pre-surgical screening.