bluuebunny/crossref_metadata_2025 huggingface dataset:
Fibrinogen And Hematocrit In Viscosity Measurements
Authors: Grotta J , Bigelow R , Olson J , Haynie D
Published: July 2018

We have studied the relative contribution of fibrinogen and hematocrit to viscosity preparatory to therapeutic reduction of viscosity in a rat model of cerebral ischemia.

Assuming Newtonian flow characteristics, we first devised a model of the interaction of these variables. Estimating the relative volume of RBCs, plasma, and visco-active proteins suspended in the plasma, assuming random collision of these components, and assuming.that viscosity is dependent only on RBCs and vaso-active proteins, we have derived the following relationship: viscosity α K1(Hct+.l)2 +K2. We then performed a linear model analysis of measured viscosity (range 1.5-12.3 cp) vs measured fibrinogen (range 0-270 mg%) and hematocrit (range 0-.45) in rat blood which was composed of either normal whole blood or mixtures of washed RBCs, defibrinogenated plasma, and known quantities of fibrinogen. Viscosity was measured in a Wells-Brookfield cone-plate micrcjjviscometer at shear rates ranging from 22.5 sec to -450 sec .

Hematocrit had a significant effect on viscosity at all shear rates measured, though this effect was greater at lower shear rates. Fibrinogen^had no effect on viscosity at shear rates above 22.5 sec-1.

These relationships are consistent with previously published data demonstrating an effect of normal concentrations of fibrinogen on viscosity only at shear rates below those accurately measured by our viscometer (i.e., less than 11 sec-1 ). We did not study fibrinogen concentrations over 300 mg%. Furthermore, there did not appear to be an interaction between fibrinogen and Hct, indicating that the absence of fibrinogen at shear rates measured in this study did not affect the properties of RBCs as they pertain to viscosity.

The accuracy of our model enables us to correct for varying Hct from animal to animal as long as measurements are performed in the Newtonian range. Presently we are testing a model of Hct and fibrinogen vs viscosity applicable to shear rates at which blood becomes non-Newtonian.
Self-Assembly Of Fibrin Monomers Measured By Quasielastic Light-Scattering
Authors: Guinnup D E , Schultz J S
Published: July 2018

Purified fibrin monomer solutions were prepared in 1 M sodium bromide by alternation of precipitation and solubilization with pH adjustments between 6.0 and 5.3. The final preparation, at pH 5.3, was checked for monodispersity and purity by quasielastic laser light-scattering and was found to have a translational diffusion coefficient of 2.14 × 10-7 cm2/sec and a radius of gyration of 286 A, in excellent agreement with published data.

The kinetics of fibrin self-assembly was monitored in-situ by measuring the autocorrelation function and mean intensity of scattered light with time after abruptly changing the pH froft 5.0 to 6.3 in 1 M NaBr. The data were most consistent with an assembly model wherein fibrin associated with itself in an overlapping staggered end to end configuration. The evolving particle size distribution of fibrin fibrils was obtained by deconvoluting the autocorrelation functions obtained over a 30 minute period and unexpectedly revealed that larger aggregates, containing greater than about 6 fibrin units, were greatly favored over smaller aggregates, dimers, trimers, etc. Also, while the rate of fibrin assembly appeared to be proportional to the square of the monomer concentration, consistent with simple aggregation theory, the probability of a collision between particles resulting in the growth of a fibril was very small.

Additional in-situ experiments under laminar shear conditions in the physiological range showed the same pattern of fibril size distribution, except that the rate of self-assembly decreased about 30% as shear increased to 1000 sec-1.
Gel Structure And Clotting Time
Authors: Blombäck B , Okada M
Published: July 2018

Ferry and Morrisson found on the basis of the mechanical and optical properties of fibrin gels that they ranged between two extreme types: fine or transparent and coarse or opaque. We have now developed a device permitting studies of flow properties of gels. The gels are formed in standardized cups (1.47×2.5 cm) which are fitted into a special holder attached to a reservoir for application of buffer solution for the flow measurement. Gels were formed by mixing thrombin or Batroxobin with fibrinogen solutions of different protein concentrations (1-4 g/1), pH (6.5-8.2) and ionic strengths (0.1-0.23). All these studies were in addition performed at different enzyme concentrations. The gels were left in the cups for complete gelation to occur (1-2 hours) and subsequently the same buffer as used for gel formation was percolated through the gels at room temperature and a constant pressure gradient. In parallell experiments the formation and optical properties of the gels formed was studied by turbidity measurement at 450 nm. After a lagtime rapid increase in turbidity occurred. The lag-time was defined as the “clotting-time”.

We found that for both enzymes the flow is related to the optical properties of the gels, being fast for the opaque and slow for transparent gels. Fast flow is thus favoured by low pH and low ionic strength. The flow rate is furthermore inversely related to the gel concentration. Of particular importance was the finding that under all conditions so far studied there was, within a wide range, a direct relationship between clotting time and flow rate. This suggests that the final structure of the gels is determined by events preceeding the gel formation. These events are enzymatic activation and alignement of activated molecules. Gel formation is therefore very likely an ordered process resembling that of crystallization. Batroxobin and thrombin gels having the same clotting times displayed different flow properties suggesting that different types of polymers are formed on release of one (FPA) and two (FPA + FPB) activation peptides, respectively.
Fibrinogen, Factor XIII And Antiplasmin In Hemostasis
Authors: Amburs C M , Ambrus J L , DeRisi D , Petrelli N J , Cohen H
Published: July 2018

Removal of large numbers of pulmonary metastases in cancer patients represents a serious problem since adequate hemostasis requires meticulous suturing and prolonged open chest procedures. For this reason, we tried to develop methods for obtaining rapid hemostasis. A double syringe system was constructed in which simultaneously we discharged from one syringe a mixture of highly concentrated human fibrinogen, platelet growth factor, antiplasmin, and Factor XIII (FS); from the second syringe we discharged thrombin and calcium chloride. After a few mm of passage through the common part of the nozzle, the mixture coated the area of pulmonary resection. In a series of experiments in dogs and stumptailed monkeys (Macaca arctoides) bleeding times of comparable pulmonary biopsies were recorded. FS alone or in association with suturing produced rapid hemostasis and as shown by follow-up histopathologic examination promoted healing without significant tissue reactions. This method proved to be superior in comparative experiments to the use of cryoprecipitate and thrombin or microfibrillar collagen preparations.

In a series of experiments, splenic injuries were induced in dogs, normally requiring splenectomy. The same three hemostatic methods described above were studied in this model. Adequate hemostasis was obtained with FS. This material appeared to be suitable to obviate the need of splenectomy and its hematologic consequences in traumatic rupture of the spleen.
Dietary Induced Changes In Arterial Thrombus Formation Is Independent From Thromboxane-Prostacyclin Balance
Authors: Hornstra G , Haddeman E , Don J A
Published: July 2018

The time needed for the complete thrombotic obstruction of a plastic aorta prothesis appeared a reliable parameter for the arterial thrombosis tendency of rats. We recently demonstrated that in this model arterial thrombus formation is regulated primarily by prostaglandin-like substances. Therefore we used this model to test the hypothesis that the arterial thrombosis tendency depends on the ratio between prothrombic thromboxane A2(TxA2) produced by activated blood platelets and antithrombotic prostacyclin (PGI2) formed by vascular tissue. Groups of 24 rats were fed diets containing 5 energy % (en%) of either sunflowerseed oil (SO, control) or hydrogenated coconut oil (essential fatty acid deficient). Four other groups received the control diet, enriched with 45en% linseed oil, cod-liver oil or hydrogenated coconut oil. After 8-10 weeks' feeding, arterial thrombosis tendency was measured in 50% of all animals. The other animals were used for quantifying the TxA2 production of collagen-activated blood platelets and the formation of PGI2 by freshly punched-out pieces of aorta. TxA2 was measured as MDA (spectrophotometry) or HHT (GC/MS). PGI2 measurements were performed with the platelet aggregation bioassay, using a standard curve obtained with synthetic PGI2. Control measurements were carried out using tissue of the same blood vessel pretreated with indomethacin. The various diets induced highly different arterial thrombosis tendencies. Platelet TxA2- and vascular PGI2-productions were significantly affected by the diet in a strikingly similar way: both were positively correlated with the arterial thrombosis tendency and the TxA2/PGI2-ratio remained essentially unchanged. Consequently, no correlation existed between this ratio and arterial thrombosis tendency. From this study it is suggested that the potency of platelets to produce TxA2 is of greater importance for arterial thrombus formation than the ability of the vessel wall to generate PGI2.
Plasma Lipoprotein Levels In Thyroid Diseases In Relation To Coronary Heart Disease
Authors: Lipinski B , Azizi F , Lipinska I , Naidu S , Mannix J E
Published: July 2018

Plasma high density lipoprotein (HDL), low density lipoprotein (LDL), very low density lipoprotein (VLDL) and total cholesterol (TC) were determined in 30 patients with thyroid dysfunction and in 58 healthy subjects. The lipoproteins were separated by means of polyacrylamide gel electrophoresis, quantitated by densitometric scanning and the results expressed as a percent of total. Percentage distribution of lipoproteins in patients with hyperthyroidism was not different from that observed in healthy subjects. In patients with hypothyroidism HDL, VLDL, LDL and TC were 17%, 16%, 66% and 320 mg/dl, and in healthy subjects were 34%, 8%, 60% and 206 mg/dl respectively. When lipoproteins were analysed in relation to the parameters of thyroid function, it was found that only HDL % values correlated well with serum thyrotropin (TSH) concentration (r=0.62) and with the free triiodothyronine (T3) index in hypothyroid patients (r=0.54). In patients who developed hypothyroidism after radioactive ablation of the thyroid, HDL % became subnormal as early as 2 months after treatment. Replacement doses of 1-thyroxine (T4) normalized plasma HDL in patients with hypothyroidism. In view of these findings, it is suggested that low levels of HDL and increased content of VLDL in plasma may play a role in the development of coronary artery disease in patients with thyroid dysfunction.
Congenital Deficiency Of Cyclo-Oxygenase In A Woman With Generalized Arteriosclerosis
Authors: Boda Z , Tamás E , Altorjay L , Pflieger Gy , Rak K
Published: July 2018

A 52-year old woman with congenital cyclooxygenase deficiency who had moderate bleeding tendency and severe generalized arteriosclerosis is reported.

Diagnosis based on the following data: secondary platelet aggregation induced by adrenaline, ADP, ristomycin, collagen, thrombin was absent; platelet completely failed to aggregate by arachidonic acid while aggregation induced by calcium ionophore A23187 was normal; no malondialdehyde formation could be detected in the platelet rich plasma /PRP/ in four different times /for exclu- sing any drug effects/. The abnormal adrenaline and ADP induced aggregation were not corrected when patient’s PRP was mixed in equal proportions with that of a normal subject ingesting aspirin.

Although our patient had been free from severe thrombotic episodes, expressed signs of generalized arteriosclerosis could be detected.

Until now only five cases of congenital deficiency of cyclo-oxygenase have been described /Malmsten et al., Weiss and Lages, Lagarde et al., Pareti et al./. However, in these patients no signs of arterial vascular diseases were mentioned.

The special importance of this new case comes from the fact that life-long deficiency of cyclooxygenase enzyme could not protect from progressive vascular disease which might prove again that chronic intake of large doses of aspirin cannot prevent arterial disorder.
Inhibition Of Cholesterol Atherosclerosis In Rabbits By Amino Imino Propene Diacetate
Authors: Weigensberg B I , Katz S , Lough J , More R H
Published: July 2018

Previous studies have shown that in vitro blockage of the ε amino group of lysine in LDL by acetoacetylation enhances LDL uptake by macrophages and liver cells and inhibits uptake of LDL by fibroblasts in tissue culture. Amino imino propene diacetate (AIPD) is a compound which is absorbed into the blood after feeding and which blocks the ε amino group of lysine in LDL for short periods of time after which the AIPD dissociates from LDL and is excreted in the urine. AIPD does not inhibit absorption of either 14C labelled or unlabelled cholesterol from the gastrointestinal tract. In a preliminary study it was found that 19 rabbits fed 300 mg crude AIPD along with 500 mg cholesterol per day in their food for 14 weeks showed 13.0 ± 3.3 (mean ± S.E.) % of their total aortic intimal surface area covered with grossly visible atherosclerotic plaques. In comparison 13 control rabbits fed 500 mg cholesterol per day in their food for 14 weeks showed 33.9 ± 6.0% of their total aortic intimal surface area covered with atherosclerotic plaques. (P< 0.01) These in vivo observations suggest that the inhibition of atherosclerosis by AIPD might be related to a reduction in the number of free lysine amino groups in LDL. In previous studies, we reported that dietary induced lysine deficiency could inhibit atherosclerosis. These and other studies suggest that blocking or reducing the number of free lysine ε, amino groups in LDL appears to enhance LDL uptake and clearance by liver and macrophage type cells and reduces LDL uptake by arterial smooth muscle cells and fibroblasts in vivo. The combination of these effects appears to offer some protection against the development of cholesterol atherosclerosis.
Suppressive Effect Of Neoratal Thymectomy On Occlusive Arterial Thrombosis In Sensitized Rats
Authors: Uzunova A
Published: July 2018

It has been found that lymphokines can induce thrombocyte aggregation and biood coagulation.

The present study investigated the development of occlusive arterial thrombosis (OAT) in both male and female Wistar rats with altered immunologic background in order to elucidate the immunologic mechanisms in the pathogenesis of OAT.

At the age of 10 weeks neonatally thymectomized rats and sham operated litter mate controls were sensitized with 500g of bovine serum albumin (BSA), injected with complete Freund’s adjuvant in a single hind footpad and a second control sham operated rats were injected only with saline. All animals were skin-tested with 30^g of BSA in saline at 7 and 14 days. Arthus reactions were read 3 to 4 hr. Both the average diameter in millimeters and thickness were recorded. Only reactions eaual and greater than 7 mm in diameter were regarded as positive.

Experimental OAT was induced when rats reached 3 months of age using a polyethylene loop inserted in the abdominal aorta. The mortality rate (MR), dry thrombus weigt (TW) and loop obstruction time (OT) served as criteria for asessment of thrombosis. According to all the criteria used there was a statistical!v significant sun- nression of the development of occlusive arterial thrombosis in the the neonatally tbvmectomized rats of either sex. The oreviouslv observed sex diferense in OAT was more pronounced.

The data presented suggested an involvement of immunologic mechanisms in the thrombogenesis in the arteries and that suitable inhibitors of the immune responses could prove to be of therapeutic value.
An Imbalance In Platelet-Vascular Prostaglandin Synthesis Induced By Homocysteine And Homocysteic Acid
Authors: Graeber Janet E , Stuart Marie J , Slott Jeffrey , Ulane Rodney E , Schulman Joseph D
Published: July 2018

Homocystinuria is a metabolic disorder characterized by serious vascular thromboses and elevated plasma levels of Homocysteine and Homocysteic Acid. The etiology of the thromboses is unknown. Platelets produce proaggregatory and prothrombotic Thromboxane A2 (TXA2) while vascular endothelium produces antiaggregatory and antithrombotic PGI2. An imbalance in production of TXA2 and/or PGI2 can lead to bleeding or thrombosis. We investigated production of platelet TXB2 (the stable end product of TXA2) and vascular 6- Keto-PGF1α (the end product of PGI2) in platelets and vascular endothelium exposed in vitro to Homocysteine and Homocysteic Acid, 0.4 to 2.0 mM. Conversion of 14C arachidonic acid (AA) to platelet TXB2 was increased in the presence of Homocysteine or Homocysteic Acid. In 8 paired experiments, mean platelet TXB2 increased to 19.4±3.5% (1SE) in the presence of Homocysteic Acid, compared to control values of 14.6±3.6% (p<0.01). Similarly, in 4 paired experiments platelet TXB2 was increased (18.7±2.3%) in the presence of Homocysteine, when compared to a mean value of 7.8±1.1% (p<0.05) in the control platelets. No differences in vascular 6-Keto-PGF1α production were observed in 6 paired experiments when vessels were incubated with either buffer, Homocysteine or Homocysteic Acid (0.73 ± .07; 0.7 ± .05; 0.76 ±.07%). These studies demonstrate that Homocysteine and Homocysteic Acid alter platelet and vascular AA metabolism by increasing production of prothrombotic platelet TXA2 without a compensatory increase in anththrombotic vascular PGI2. Such an imbalance may explain the marked thrombotic tendency seen in patients with homocystinuria. It also provides evidence for the use of thromboxane synthetase inhibitors in this disorder.
Coagulation And Hem0Rhe0L0Gy In Cerebrovascular Disease. Differences Reated To Angiographic Findings And Sex
Authors: Coccheri S , Palareti G , Andreoli A , Poggi M
Published: July 2018

A series of 102 cerebrovascular patients (CVP) investigated with aortic arch angiography, carotidography and CAT scan, were classified clinically in STROKE (55) and TIA (47) and pathologically as having (IAL, 51) or having no (NIAL, 51) identifiable arterial lesions. Coagulation and hemorheologic tests were performed at least 3 months after STROKE or 1 month after a TIA episode. All CVP as compared to controls (84) had significantly higher fibrinogen (Fg), F VIII AHF and RAg, blood (BV) and plasma (PV) viscosity, and poorer erythrocyte deformability (ED, as filtered erythrocyte volume, FEV), but no differences in euglobulin lysis time (ELT) even after venostasis (ELTV) and in circulating platelet aggregates (CPA, as 1/PAR). IAL vs NIAL CVP had higher Fg (mg% 285±74 vs 241±64; p<0.005); a trend to higher BV (cp 4.64±0.5 vs 4.53±0.6) and PV (cp 1.63±0.15 vs 1.58±0.15), but no differences in other parameters. In the subgroup IAL-STROKE more CPA (1/PAR 121±18) were found vs IAL-TIA (1/PAR 104±13; p<0.05). When compared to sex-matched controls CV males had more Fg (269±36 vs 220±38; p<0.01), higher BV (p<0.05) and PV (1.61±0.16 vs 1.45±0.13; p<0.001), and poorer ED (FEV ml/min 7.41±2.8 vs 10.6±3.2; p<0.005). CPA were higher in CV males than in CV females (1/PAR 122±31 vs 96±36, p<0.05). Conversely, CV females differed from their controls only for a higher PV (p<0.05). This study points out that most of the parameters considered, are especially altered in CV males rather than in CV females, thus suggesting sex-related differences in response to drugs acting on haemostasis and rheology in CV diseases.
Diagnosis Of Deep Vein Thrombosis - “The Case for NonInvasive Testing”
Authors: Hull R , Hirsh J
Published: July 2018

Ascending venography, although the diagnostic standard for deep vein thrombosis (DVT), has important clinical pitfalls and shortcomings. It is invasive and thus not readily repeated: its use is associated with significant discomfort in many patients and in 3-4% of patients post-veno- graphic phlebitis is induced. A high degree of technical and interpretive skill is required and in up to 20% of patients routine ascending venography fails to visualize the external and common iliac veins. In many hospitals, outpatient access is not readily available necessitating admission to hospital for elective venography. Non-invasive testing with impedance plethysmography (IPG) is gaining increasing acceptance and use because it is objective, versatile and free of morbidity. IPG is sensitive and specific for symptomatic proximal DVT, but has the potential limitation that it is insensitive to calf DVT. Because of this, two different non-invasive approaches are currently advocated: a) serial IPG’s to detect calf vein thrombi which extend proximally (advocates of this approach suggest that calf DVT rarely lead to symptomatic pulmonary emboli unless proximal extension occurs) and b) addition of leg scanning to detect calf DVT. The effectiveness of serial IPG’s is uncertain and to resolve this issue we are currently performing a randomized trial. Multiple large studies however demonstrate that because of both high sensitivity and specificity, the combined approach of IPG and leg scanning provides a replacement for venography in the majority of symptomatic patients. Furthermore, the safety of witholding anticoagulant therapy in patients negative by combined IPG and leg scanning has been confirmed by long-term follow-up. Combined IPG and leg scanning is more cost-effective than elective venography because these non-invasive tests are readily performed in the emergency room or clinic, thus preventing unnecessary admission to hospital of patients with clinically suspected DVT who are negative by testing.
Complex Formation Of Platelet Glycoproteins IIB And IIIA With Fibrinogen
Authors: Nachman R , Leung L
Published: July 2018

Platelet membrane glycoproteins IIb and IIIa were isolated and purified from human platelet membranes using lentil lectin affinity chromatography and electrophoretic elution from SDS-poly-acrylamide gels. Two dimensional immunoelectrophoresis of a mixture of the purified proteins against monospecific antisera showed antigenic uniqueness of the separate polypeptides. Computerized analysis of autoradiographs of two dimensional tryptic 125I peptide maps revealed that the two glycoproteins had completely different structures. Thus human platelet membrane glycoproteins IIb and IIIa are separate molecular entities. Monospecific anti-GPIIb and anti-GPIIIa were utilized in an enzyme linked immunosorbent assay system to demonstrate complex formation of the platelet glycoproteins with purified human fibrinogen. The formation of this GPIIb-GPIIIa fibrinogen complex is calcium dependent, fibrinogen specific, saturable, and inhibited by specific amino sugars and amino acids. These observations suggest that the GPIIb-GPIIIa macromolecular complex on the platelet surface acts under the proper physiologic circumstances as the fibrinogen binding site required for normal platelet aggregation.
Influence Of High Molecular Weight Factor VIII On The Expression Of Factor VIII Procoagulant Activity
Authors: Muntean W , Hathaway W E , Montgomery R R
Published: July 2018

The relationship of the high molecular weight (HMW) moiety and low molecular weight (LMW) moiety of factor VIII in expressing procoagulant activity (VIII C) was studied. LMW VIII C was prepared by immunoadsorbent chromatography; HMW VIII was prepared by chromatographing hemophilic cryo- precipitate in 4% agarose. The LMW VIII C obtained by immunoadsorbent chromatography gave higher VIII C values when tested in the one stage partial thromboplastin time (PTT) system using von Willebrand’s disease plasma as substrate than using hemophilic plasma as substrate. This finding was shown to be due to the VIII related antigen (VIIIR:Ag) in the substrate plasmas. When the VIIIR:Ag was removed from the hemophilic substrate plasma by immuno-adsorption, the VIII C values obtained for the LMW VIII C were higher. Also, adding purified HMW VIII to the von Willebrand’s disease substrate plasma resulted in lower VIII C values for the LMW VIII C in the PTT system.

When the LMW VIII C was tested in the two stage assay, all VIII C was adsorbed to A1(0H)3. The adsorption of the LMW VIII C was prevented by mixing it with hemophilic plasma. From normal undiluted plasma only 5-21% of VIII C and no VIII related antigen were adsorbed to A1(OH)3, but after activation of the factor VIII of normal plasma by small amounts of thrombin, most of the VIII C was adsorbed. No VIII related antigen was adsorbed after activation.

Nevertheless, when unadsorbed LMW VIII C was assayed by the two stage method both with and without HMW VIII or VIIIR:Ag, the results were the same.

Our studies suggest that VIIIR:Ag prevents to some extent the activation of LMW VIII C. LMW VIII C that is not bound or protected by VIIIRiAg is adsorbed from plasma by A1(0H)3. These findings may help explain the differences for VIII C found in some patients and certain clinical circumstances with the one and two stage assays.
Structural Studies On The Functional Heterogeneity Of Von Willebrand Protein Polymers
Authors: Martin S Eric , Marder Victor J , Francis Charles W , Barlow Grant H
Published: July 2018

Human von Willebrand protein purified from cryoprecipitate was separated by gel elution and sucrose gradient ultracentrifugation into groups of polymers of different size, ranging from a molecular weight greater than 10 × 10-6 to a minimum of 2.4 × 10-6. After disulfide bond reduction, all polymers showed a major band of 208,000 molecular weight with about 1% of the protein having lower molecular weights of 197,000, 174,000 and 154,000. Major and minor moieties were recovered from immunoprecipitates obtained with monospecific anti-von Willebrand protein antibody. The ristocetin cofactor activity of the different polymers showed increasing specific activity with increasing molecular weight, whether measured relative to von Willebrand antigen value or content of the 208,000 molecular weight subunit chain. This difference in specific activity was particularly evident when comparing groups of molecular weight greater than 10 × 106 with those of molecular weight less than 5 × 106. There was no difference in the content of the minor reduced bands in each polymer, no difference in carbohydrate concentration or susceptibility to neuraminidase or galactose oxidase, and no difference in the pattern of tryptic degradation or function of the 116,000 molecular weight tryptic remnant that retains ristocetin cofactor activity. The disulfide bond organization of the larger polymers appears to differ from that of the smaller polymers inasmuch as partially reduced polymers obtained from the high specific activity group expressed more ristocetin cofactor activity than unreduced polymers of similar size present in the low specific activity group. Apparently, optimal interaction of the von Willebrand polymers with platelets is dictated not only by size but also by tertiary structure as shaped by disulfide bond organization.
Comparison Of Naturally Occurring And CaCl2-Separated Canine Factor VIII-Coagulants Free Of FACTOR VIII-Related Antigen
Authors: Benson R E , Dodds W J
Published: July 2018

Previous studies demonstrated that plasma from dogs homozygous for von Willebrand’s disease (VWD) without detectable factor VUI-related antigen (VIII:RAg) contained moderate levels of a stable factor VIH-coagulant (VIII:C), which had lower apparent molecular weight and combined with the VIII:RAg of canine hemophilic plasma. We have now compared this VWD-VIII:C to the CaCl2-separated form of VIII:C prepared from normal canine factor VIII. The VWD plasma was fractionated at room temperature by 6% agarose gel filtration in 0.15M NaCl and 0.24M CaCl2 buffers, pH 7.25; the VIII:C eluted in each buffer with a relative elution volume (Ve/Vo) of 1.4. Isolated lower molecular weight VIII:C stabilized with albumin (5 mg/ml) and dialysed free of Ca++ was prepared from normal purified canine factor VIII by elution in 0.24M CaCl2 buffer. This CaCl2~separated VIII:C was rechromatographed in both buffers as above and had a Ve/Vo of 1.9. When the VWD and CaCl2 forms of VIII:C were each combined with canine hemophilic plasma and gel filtered in 0.15M NaCl buffer, the VIII:C’s now appeared at the void volume. These mixtures with 1/10 volume 2.4M CaCl2 added before or after combination were also analyzed in 0.24M CaCl2 buffer. The CaCl2-VIII:C eluted with a Ve/Vo of 1.9 regardless of the order of 2.4M CaCl2 addition, but the VWD-VIII:C eluted with Ve/Vo’s of 1.4 when CaCl2 was added before mixing and 1.9 when added afterwards.

Thus, when mixed with hemophilic plasmas both the CaCl2 and VWD forms of VIII:C exhibited similar combination and separation characteristics. However, the finding that the CaCl2-VIII:C is of apparent lower molecular weight than the VWD form suggests that the CaCl2-separated VIII:C is different from the naturally occurring form of VWD (VIII: RAg-free) VIII:C.
Factor VIII/Von Willebrand Factor Protein Sensitivity Of Periodic Acid Schiff Stain To Carbohydrate Deficiency
Authors: Gralnick H , Jackson G , Williams S , Cregger E
Published: July 2018

Carbohydrate (C) deficiency of the factor VUI/von Willebrand factor (f. VIII/vWf) protein has been implicated as one of the molecular defects of von Willebrand’s disease. The periodic acid Schiff (PAS) reaction in polyacrylamide gel electrophoresis (PAGE) has been used as a screening test for C deficiency. To ascertain the sensitivity of this reaction to the C content of the f. VIII/vWf protein we have purified the normal protein and compared the Coomassie to PAS staining reaction of the subunit of the intact, asialo (neuraminidase-treated), asialo-agalacto (neuraminidase and β-galactosidase-treated) derivatives and a fourth derivative which was treated with a mixed glycosidase which included neuraminidase, β-galactosidase and N-acetyl-glucosaminidase. Gels were run simultaneously, one stained with Coomassie blue and the other with PAS and at various time intervals after staining the gels were scanned.

Studies of the intact f. VIII/vWf protein revealed that the PAS and Coomassie stains could be compared from 24 hours up to 7 days with no significant changes. The desialated f. VIII/vWf protein had insignificant differences in the PAS stain compared to normal (range 81-108% of intact). However, the asialo-agalacto derivative (36% of intact) and the f. VIII/vWf treated with the mixed glycosidase (24% of intact) had significantly reduced PAS staining in relation to normal. C analysis of the different f. VIII/vWf proteins revealed that removal of the sialic acid reduced the C 30% while in the asialo-agalacto the C was reduced 47% and in the mixed glycosidase-treated material the C was reduced 78%. The PAS stain is not very sensitive to the sialic acid content of the f. VIII/vWf protein and reduction of 30% of the C does not affect the PAS stain. Since the PAS stain is relatively insensitive to significant carbohydrate deficiencies of the f. VIII/vWf protein, the use of the PAS stain in PAGE may not be an adequate test to ascertain C content of the f. VIII/vWf protein.
A family with factor XII deficiency: a family study suggesting that factor XII levels are determined by the presence of 3 codominant alleles with variable penetrance
Authors: Roberts D , Hayne O , Fraser E
Published: July 2018

We studied 15 members, in 3 generations of a family in whom the propositus had complete factor XII deficiency. The parents had factor XII levels of 59% and 62%. Three of the siblings had factor XII levels of less than 1%. Three more had factor XII levels of (30±5%). In 3 the levels ranged from (60-87%) and the remaining 2 siblings had levels exceeding 100%. The propositus with a level of less than 1% gave rise to an offspring with a level of 48%.

We propose that the gene controlling factor XII production consists of 3 separate alleles expressed codominantly: A - normal, B - low, O - absent. Members of the family with 100% factor XII activity have genotype AA; those with less than 1% activity have genotype OO; subjects with levels of 30% have genotype BO, and those with levels of 60% have genotype AO. We feel our study sheds new light on the inheritance of factor XII and the wide variation of levels in the general population.
Native factor XII (FX11) requires proteolytic cleavage for activation
Authors: Silverberg M , Kaplan A P
Published: July 2018

It has been proposed that FX11 zymogen can cleave pre- kallikrein without prior conversion into one of its proteo- lytically cleaved forms FXlla or FXllf. We treated FX11 with pro-phe-arg-chloromethyl ketone (PPACMK) to remove all measurable amidolytic activity due to traces of FXlla or FXllf. Prekallikrein (PK) and IgG were similarly treated to remove all traces of kallikrein. FX11 was diluted to 0.016 μg/ml, using 3.8mg/ml IgG as the diluent, and mixed 1:1 with 215μg/ml PK. The rate of generation of kallikrein activity was neglible to begin with but increased with time to approach the rate of activation obtained with FXlla or FXllf at equivalent concentrations. When 10μg/ml dextran sulphate was present the rate of PK activation was maximal from the beginning. This experiment was repeated in the presence of a specific inhibitor of activated FX11 derived from corn; O.lmg/ml inhibitor prevented the activation of PK. This indicates that the PK activator is the same species that reacts with the inhibitor, namely FXlla or FXllf. In another experiment, 1.67μg/ml of PPACMK-treated FX11 and 2.6 μg/ml PPACMK-treated PK were mixed in glass cuvettes containing 500yM chromogenic substrates specific for either FXlla or kallikrein. The resulting curves of absorbance vs time showed that both FX11 and PK became activated in accelerating reactions after short lag phases; corn inhibitor blocked this reaction too. Thus the rapid activation and the lack of a lag phase seen in the experiment with dextran sulphate was not due to a substrate-induced formation of an active site in FX11 zymogen but was a result of the very rapid activation of surface bound FX11 by traces of kallikrein. The kallikrein may have been generated by traces of FXlla which are always present even after treatment of FX11 with inhibitors. This amount of FXlla may also be present in vivo and provides a potential mechanism for the initiation of contact activation.
Relative Importance Of Plasma Protease Inhibitors In The Inactivation Of Kallikrein In Human Plasma
Authors: Schapira M , Scott C F , Colman R W
Published: July 2018

Human plasma contains several inhibitors of plasma kallikrein (KAL), including C1-inhibitor (C1INH), α2-macro- globulin (α2M), antithrombin III (ATIII), and α1-antitrypsin (αlAT). Studies in purified systems have allowed us to quantitate the kinetic constants of each isolated inhibitor. We also have demonstrated that high molecular weight kininogen (HMWK) is an important regulator since it decreases the inactivation rate of KAL by all inhibitors except α2M. When purified inhibitors and HMWK are present at plasma concentrations, it can be calculated that C1INH, α2M, ATIII and α1AT account respectively for 49%, 49%, 0.8% and 0.2% of the KAL inhibitory activity. To assess if this prediction derived from purified system adequately describes the inhibition of KAL under more physiological conditions, we incubated KAL with normal human plasma (NHP), C1INH-deficient plasma (C1INH-D), and α2M-deficient plasma (α2M-D). KAL activity_was assessed using H-D-Pro-Phe-Arg- Nan as a substrate. C1INH-D was obtained from a patient with hereditary angioedema. C1INH in C1INH-D was 15% of the normal value as assessed by radial immunodiffusion. α2M-D was obtained by selective and complete inactivation of α2M by methylamine. The pseudo-first-order rate constants for the inactivation of_KAL by NHP, C1INH-D, α2M-D, and plasma deficient in both C1INH and α2M were respectively 8.8, 5.0, 5.5, and 1.8 S-1(×l03). Therefore, C1INH accounted for 63% of the observed inhibition, α2M for 35%, and all the other inhibitors for 2%. When these values were corrected for the concentration of HMWK, C1INH accounted for 47% of the inhibition, α2M for 51%, and all the other inhibitors for <2%. Thus, there is an excellent agreement between the results obtained when KAL is inhibited in purified systems and those obtained when it is inhibited in plasma. Moreover, these results indicate that C1INH and α2M are the only important inhibitors of KAL in NHP.
In Vivo Thromboscintigraphy With Indium-111-Labeled Platelets In A Dog-Catheter Model
Authors: Price D C , Lipton M J , Hartmeyer J A , Prager R J
Published: July 2018

Autologous platelets from mongrel dogs have been labeled with 200-400 μCi Indium-111 complexed to oxine using the technique of Thakur, McAfee and others. Intraarterial thrombogenesis was studied in vivo by advancing a polyethylene angiographic catheter from a femoral into a carotid artery, then serially imaging the catheter over periods of 0.5-3 hours by scintillation camera with interfaced computer. Quantitative uptake was derived from computer processing of the studies, and compared with in vitro In-111 counts and clot weight obtained at various times by catheter excision. Labeled platelets were injected prior to or at periods of 2 or 24 hours after catheter insertion in order to evaluate both forming and preformed thrombus. Platelet In-111 radioactivity was found to peak at 30-80 minutes in newly forming thrombus and to fall thereafter. In vitro In-111 activity correlated well with wet clot weight.

In-111 (% inj. dose) = 0.00209 × (mgm clot) + 0.00091 (r = 0.882) (n = 24)

In vivo correlation was also linear, but with a broader scatter of data points. At the peak, In-111 uptake was 0.06044). 019% of i.d. per cm of catheter by in vitro measurement, and clot weight was 27.9±5.6 mgm per cm. Preformed thrombus picked up substantially less of the label (2 hr: 0.017±0.011% i.d./cm; 24 hr: 0.00245).001% i.d./cm) (n=6) and was not imageable in vivo. The study documents the effectiveness of this new platelet radiolabel for quantitative in vivo scintigraphy of newly forming thrombus, which can be useful for comparison of different anti-thrombogenic regimens and different biomaterials. It also indicates, however, the problems that can be anticipated in attempting to image established thrombus in vivo.
Rates Of Fibrin Growth As A Predictor Of Acute Myocardial Infarction. A Prospective Blind Study
Authors: Joe Baughman D , Kostis J B
Published: July 2018

A blind prospective coagulation profile was performed on 147 patients with coronary artery disease (CAD). Theprofile consisted of Prothrombin time (PT), the maximum rate of fibrin production (turbidity) when measuring the PT (PT Vmax), Activated Partial Thromboplastin time (APTT), APTT Vmax, Thrombin time (TT), TT Vmax, Fibrinogen (F), plasma and serum Antithrombin III (At-III), Lysis Time (LT) and Lipoproteins (LP). All clotting times and turbidities were measured using a BioData CP-7; At-III was measured as the loss of thrombin clotting activity; F was measured as the total thrombin clottable protein; LT was measured by a modified CLUE test; LP was measured as a heparin-Mg produced turbidity.

During 41 months of follow-up, 12 patients developed a new myocardial infarction (MI), and had significantly higher APTT Vmax (7.46 ± .30U vs. 6.21 ± .09U, p=.0001); PT Vmax (7.83 ± .27U vs. 6.46 ± .10U, p=.0002); TT Vmax (5.33 ± .32U vs. 4.20 ± LOU, p=.0014); and F (339.6 ± 12.2 mg/dl vs. 292.76 ± 4.6 mg/dl, p=.003) than patients who did not. Out of 37 patients with the highest PT Vmax (upper 25%), 27% developed acute MI, while only 2 (1.8%) of the remaining 110 developed MI, giving a risk ratio of 15.4, p=.0001. Similarly, 9 out of 12 infarctions occurred in the upper 25% of APTT Vmax, giving a risk ratio of 8.9, p=0.0004. MI was also predicted by TT Vmax with a risk ratio of 5.9 (p=0.0004) and by fibrinogen with a risk ratio of 4.8 (p=0.0018). No relationship between MI and the other coagulation tests was noted. Thus, 1. rates of fibrin growth may predict the occurrence of MI in CAD, and 2. soluble coagulation parameters are important in the pathogenesis of acute myocardial infarction.
Interventional Radiography In Renal Artery Thrombosis
Authors: Zajko A , Solomon M P , Ring E J , Grossman R A , Barker C F , Perloff L J
Published: July 2018

Recent reports of successful revascularization of kidneys sustaining prolonged warm ischemia secondary to acute renal artery occlusion prompted the following clinical trial in a renal allograft recipient: During treatment for a mild rejection reaction 19 days after receiving an HLA identical renal allograft, a 30-year old male suddenly developed oliguria with serum creatinine and BUN rising from 2.0-3.4 and 35-61 mg/dl respectively. DTPA technetium and I131 hip- puran scans revealed functional activity and flow limited to the upper pole of the allograft. Arteriography showed multiple intrarenal emboli, a stenotic main renal artery distal to the internal iliac anastomosis, and complete occlusion of the lower pole artery. After transluminal dilatation of the stenosis with a 4 mm balloon catheter, the anastomosis was easily trasversed with coaxial catheters for infusion of streptokinase (5000U/hr) and heparin (800U/hr). Repeat arteriography 12 hours later showed a decrease in the amount of thrombosis. Streptokinase infusion rate was increased to 7500U/hr, and heparin was administered by continuous systemic infusion to maintain the PTT at 45-50. By manipulation of the catheters, thrombi in the major renal artery branches were maneuvered into the already occluded lower pole artery. At 36 hours arteriography revealed reopening of many of the distal occlusions. During this time fibrin degradation products were 10 μg/ml, thrombin time was 64 seconds and fibrinogen level was 235 mg/dl. Although the patient required four weeks of dialysis the urine output was maintained at >1000 ml/day, suggesting polyuric acute tubular necrosis. Five weeks after the occlusive episode, the main renal and upper pole arteries of the allograft were patent and serum creatinine was 3.5 without dialysis.

The previously poor results following acute renal artery occlusion in the renal allograft recipient may be improved with early interventional arteriography. This may provide marginal perfusion until surging 1 intervention ensues.
Cardiovascular Function Following Surgical Stimulation Of Pulmonary Prostacyclin Synthesis
Authors: Hechtman H B , Krausz M M , Utsunomiya T , Levine L , Shepro D
Published: July 2018

Surgery initiates local wound hemostasis and a systemic reaction which we hypothesize protects against intravascular coagulopathy. This study examines release and systemic effects of prostacyclin (PGI2) and thromboxane (Tx) in response to surgery. In 10 dogs laparotomy or thoracotomy was followed by a rise in arterial concentrations of 6- keto-PGF1α , the stable metabolite of PGI2 from 0.02 to 0.26 ng/ml (p < 0.005), a value higher than pulmonary arterial levels (p < 0.05). TxB2, the stable metabolite of TxA2 , was unchanged. Cardiac output (CO) rose from 3.46 to 4.06 L/min (p < 0.05) whereas mean arterial pressure (MAP) fell from 142 to 122 mm Hg (p < 0.03). ADP induced platelet aggregation decreased from 491 to 28% (p < 0.001). Indomethacin, 5 mg/kg IV, in 5 dogs prevented the rise of 6- keto-PGF1α secondary to surgery, and levels were now higher in mixed venous than arterial blood (p < 0.03). There were no changes in CO, MAP or platelet aggregation. During abdominal aortic aneurysmectomy (14 patients), 6-keto-PGF1α increased from 0.04 to 0.95 qg/ml (p < 0.01) 30 min after incision, but before transfusion or aortic clamping; TxB2 did not change. Cardiac index (Cl) rose from 2.72 to 3.04 L/min.m2 (p < 0.03) and MAP fell from 100 to 88 mm Hg (p < 0.01). Platelet aggregation decreased from 55% to 40% (p < 0.001) and platelet counts decreased to 166,000/mm3, 24 h postop (p < 0.01). In 9 patients given aspirin (650 mg) by mouth 12 h preoperatively the rise in 6-keto-PGF1α and Cl, and fall in MAP and platelet aggregation were prevented. Mean platelet counts were 107,800/mm3, 24 h postop, which was lower than observed in patients not given aspirin (p < 0.005). Surgical trauma stimulates the lungs to secrete PGI2 , which has systemic hemodynamic and hematologic consequences.
Increased Circulating 6-OXO-PGF1α During Angiotensin II Infusion In Man
Authors: Silberbauer K , Sinzinger H
Published: July 2018

Angiotensin II(A II) is able to stimulate phospholipase A2. This enzyme initiates the arachidonic acid cascade. Previously we have shown that All enhances the PGI2 release from rat aortic rings and rat kidneys.

We, therefore, wondered if All infusion in man might influence the plasma concentrations of 6-OXO-PGF1α (RIA in unextracted plasma), the stable metabolite derived by nonenzymatic conversion from PGI2. Fourteen patients with normal blood pressure, who were tested to assess left ventricular function by radionuclide angiography, received All (2-l,5μg/min for 15 minutes i.v.). During the rise of blood pressure (x:125/80mmHg to 157/102mmHg)6-OXO-PGF1α increased significantly (2p<0.01). Fifteen minutes after All termination the blood pressure returned to prevalues(x:125/82mmHg) and 6-OXO-PGF1α dropped. In one patient saralasin, a competitive inhibitor of All, had no direct effect on 6-0X0-PGF1α (0.1μg,1μg and 10μg saralasin/kg b.w./min, each dosage was infused for 15 minutes).

The released vasodilator PGI2, as measured by 6-OXO-PGF1α generated in the vessel wall and in the kidney, might antagonize the vasoconstriction caused by AII.
The Effect Of Platelet Activation On Internal pH
Authors: Wencel June D , Feinberg Harold
Published: July 2018

The intracellular pH of human blood platelets was investigated by studying the distribution of a radiolabeled. pH sensitive probe, 14C-methylamine (MEA). In a typical donor, MEA distribution indicated an internal pH (pHi) of 7.17 ± 0.12 SEM. This value remained constant when the platelets were challenged by the addition of external acid or base over a pH range of 6.5 to 8.0, indicating a considerable internal buffering capacity. ^lp-nUCTear magnetic resonance verified MEA distribution by yielding similar values. The possibility that pHi changes during activation or serves as an activation trigger was investigated. Stimulation by ADP (1 x 10-5M) resulted in a significant (p<.025) acidification of the platelet cytosol. This acidification was blocked by preincubation with 2-deoxy-glucose (20 mM) while aggregation was not. Additionally, no significant acidification was observed after stimulation by epinephrine (5 x 10-5m) even though a normal aggregation response was elicited. These results suggest that cytosolic acidification is not a prerequisite to aggregation and that at least as measured by MEA distribution, pHi shifts apparently do not have a primary or triggering role in ADP or EPI induced aggregation but may reflect secondary changes due to the stimulation of metabolism.