Effects of high-molecular-weight cryoprotectants on platelets and the coagulation system

The objective of this study is to examine the effects of the most widely used high-molecular-weight cryoprotectants on the coagulation system. Dextran, hydryoxyethyl starch (HES), polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), and albumin were added at different concentrations in the range between 0.01-1% (w/v) to solvent/detergent-treated plasma. Using a STA/STA Compact coagulation analyzer the following clotting tests were performed: prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), Factor V, and Factor VIII percentage of activity. PVP and PEG caused a significant increase in APTT, a decrease in Factor VIII percentage of activity, and a slight decrease in TT, while PT and Factor V percentage of activity remained unchanged. Dextran, HES, and albumin did not effect the clotting tests. The effect of high-molecular-weight cryoprotectants on platelets was assessed by platelet-induced clot retraction (PICR) and aggregation with thrombin and agglutination with ristocetin. Platelet aggregation and agglutination were unaffected by all cryoprotectants tested; however, PICR was significantly reduced in the presence of PVP or PEG. Possible mechanisms by which PVP and PEG interfere with the coagulation system are discussed. We also raise issues concerning the development of one-step blood cryopreservation techniques which do not require cryoprotectant removal prior to transfusion.     

Circadian rhythms of blood clotting time and coagulation factors II, VII, IX and X in rats

The 24-hr variations in clotting times and vitamin K-dependent blood coagulation factors were studied in rats kept on a 12-hr light-dark cycle (light on: 0600-1800 hours). Clotting times were determined under a binocular microscope by measuring the time required for the formation of the first fibrin thread. Factors II, VII and X were analyzed by the prothrombin test while the factor IX was quantified using the activated partial thromboplastin time assay. Results indicated that the clotting times were significantly longer during the dark (activity) period with a peak at 1:00 and a trough at 17:00. Similarly, a variation was found in factor activity levels: prothrombin (II), factor VII and factor X had higher activities during the light span (rest period). The highest activities found at 13:00 and 09:00 were statistically different from the minimum activity levels obtained at 21:00. Factor IX did not show a significant circadian variation.     

The effect of cyanate on the clotting proteins and platelet function.

Cyanate reacts with unchanged amino groups of various proteins in a specific irreversible carbamylation reaction. The effect of this molecule on the clotting process and the effects of carbamylation on the clotting proteins and platelet functions were investigated in vitro. An immediate effect on the clotting proteins, not related to pH, was seen in the screening tests prothrombin time, partial thromboplastin time and thrombin time at the highest concentration (100 mM), to a lesser degree at the lower concentration (10 mM). These changes reflected decreases of 19 and 36% respectively in Factor V and X activity, an inhibition of 63-75% of Factors VII, IX, X and XI activity, and 80% inhibition of thrombin activity. The inhibitory changes of carbamylation increased with time. No changes were seen in the activity of Factors I and VIII. Platelet function studies revealed no inhibition of Factor III release; aggregation was abnormal only at high concentrations with epinephrine and collagen induction and partially reversible by resuspension in normal plasma.     

Evaluation of the efficacy and safety of rivaroxaban using a computer model for blood coagulation

Rivaroxaban is an oral, direct Factor Xa inhibitor approved in the European Union and several other countries for the prevention of venous thromboembolism in adult patients undergoing elective hip or knee replacement surgery and is in advanced clinical development for the treatment of thromboembolic disorders. Its mechanism of action is antithrombin independent and differs from that of other anticoagulants, such as warfarin (a vitamin K antagonist), enoxaparin (an indirect thrombin/Factor Xa inhibitor) and dabigatran (a direct thrombin inhibitor). A blood coagulation computer model has been developed, based on several published models and preclinical and clinical data. Unlike previous models, the current model takes into account both the intrinsic and extrinsic pathways of the coagulation cascade, and possesses some unique features, including a blood flow component and a portfolio of drug action mechanisms. This study aimed to use the model to compare the mechanism of action of rivaroxaban with that of warfarin, and to evaluate the efficacy and safety of different rivaroxaban doses with other anticoagulants included in the model. Rather than reproducing known standard clinical measurements, such as the prothrombin time and activated partial thromboplastin time clotting tests, the anticoagulant benchmarking was based on a simulation of physiologically plausible clotting scenarios. Compared with warfarin, rivaroxaban showed a favourable sensitivity for tissue factor concentration inducing clotting, and a steep concentration-effect relationship, rapidly flattening towards higher inhibitor concentrations, both suggesting a broad therapeutic window. The predicted dosing window is highly accordant with the final dose recommendation based upon extensive clinical studies.     

Blood coagulation studies in guineapigs (Cavia porcellus)

Blood coagulation studies were performed on 45 healthy, adult guinea pigs. Additionally thrombelastograms of 30 animals were recorded. Guineapigs revealed short partial thromboplastin times and euglobulin lysis times, but long prothrombin times and thrombin times. Fibrinogen values were within the range of human normal values. Biphasic ADP-induced aggregation of platelets, as occurs in man, was found in 29% of the animals. Short r (reaction time until the beginning of clot formation) and k times (time from the beginning of clot formation until an amplitude of 20 mm) of their thrombelastograms indicate, that whole blood clotting is enhanced in guineapigs. Higher maximum amplitudes in this species suggest a stronger clot stability than in man.     

Human thrombins. Production, evaluation, and properties of alpha-thrombin.

Human alpha-thrombin, the thromboplastin activation product of prothrombin with high clotting and esterase activity, was produced from Cohn Fraction III paste. The procedure started with 0.4 to 3.2 kg of frozen paste and was completed in 2 or 3 days. Some 23 g of thrombin were recorded for 65 quantitated preparations made from 11 lots of Fraction III paste. These preparations were obtained at protein concentrations of 3.9 +/- 1.3 mg/ml with a yield of 340 +/- 110 mg/kg of paste, which represented 48 +/- 14% of the clotting potential extracted as prothrombin. They had specific clotting activities of 2.8 +/- 0.4 U.S. (NIH) units/microng of protein and titrated to 88 +/- 8% active with p-nitrophenyl-p'-guanidinobenzoate (NPGB). Those (N - 29) examined by labeling with [14C]diisopropyl phosphorofluoridate (iPr2P-F) and electrophoresing in sodium dodecyl sulfate (SDS)-polyacrylamide gels were found to contain only (N = 4) or predominantly alpha-thrombin (97 +/- 3%) and corresponding amounts of ists degradation product, beta-thrombin (2.6 +/- 3.1%). No plasmin(ogen), prothrombin complex factors (II, VII, IX, IXalpha, X, Xalpha), or prothrombin fragments were detected in representative preparations. As produced in 0.75 M NaCl, pH approximately 6, thrombin was stable for approximately 1 week at 4 degrees and for greater than 1 year at less than or equal to 50 degrees; freeze-dried thrombin stored at 4 degrees for greater than 1 year displayed stable clotting activity and no vial to vial variation, permitting its use for reference purposes. Human thrombin generated by Taipan snake venom activation was compared with that produced by rapid thromboplastin activation: after treatment with [14C]iPr2P-F, greater than 95% of the label in both thrombins migrated at the same rate during electrophoresis in SDS; identical pairs of NH2-terminal residues were released in three consecutive Edman degradation cycles.     

Prothrombin activation by immobilized thrombin]

The ability of thrombin, immobilized on BrCN-activated Sepharose 4B, to split prothrombin, was studied. Immobilized thrombin retained up to 70% of its esterase activity and about 5% of its coagulating activity; it was also found to induce partial proteolysis of prothrombin. Two products of prothrombin degradation isolated, i.e. P1 (m. w. 50.000-52.000) and P2 (m. w. 22.000-24.000), did not show either the thrombin or the prothrombin activities. P1 was converted into thrombin under the action of tripsin or Factor Xa. The rate of conversion was considerably increased after addition of Factor V, thromboplastin and Ca2+ ions. Intravenous administration of P1 to rats resulted in changes in the coagulating system of blood, which may be probably indicative of the stimulation of the anticoagulating system. P2 possessed no thrombogenic activity.     

A family with heterozygous factor X Friuli defect outside Friuli

Three members of the same family were found to have a clotting defect consistent with the diagnosis of heterozygous factor X Friuli disorder. The main features of the defect were a mild prolongation of prothrombin time and partial thromboplastin time, but a normal Stypven-Cephalin clotting time. Factor X activity was 40-50% of normal using tissue thromboplastin, but was perfectly normal using Russell's viper venom and cephalin. Using chromogenic substrate S-2222 the level was 30% of normal. Immunologically, factor X was normal. Bleeding manifestations were mild if any. The hereditary pattern was autosomal. The family comes from an area far away from Friuli and represents the first example of factor X Friuli discovered outside the Friuli.     

Simultaneous determination of zymogen activation time and zymogen level using chromogenic substrates in blood coagulation analysis

The amidolytic activities of plasma generated by means of thromboplastin and Ca++, on the one hand, and by means of partial thromboplastin, a contact activator and Ca++, on the other hand, were determined using synthetic, chromogenic factor Xa substrates with low affinity for thrombin (CH3SO2-D-Leu-Gly-Arg-pNA and CH3SO2-D-Nleu-Gly-Arg-pNA). In this way, the activation process by splitting off the p-nitroaniline was followed. Besides the summary detection of factor Xa was obtained after addition of hirudin. During preincubation with partial thromboplastin and contact acti (Actin) in Ca++-free medium, an amidolytic activity so far unidentified was generated that renders evaluation of the activation process difficult. In the test system with partial thromboplastin, factor Xa could not be determined and the thrombin-like activity that can be inhibited by hirudin did not correspond to the amount of prothrombin present in plasma. In contrast, activation of factor X and prothrombin by thromboplastin and Ca++ could be followed and the content of the two zymogenes could be detected simultaneously. In general, under optimized reaction conditions, automated systems might be developed that would provide additional diagnostic information about determination of clotting time, on the one hand, and about quantitative determination of zymogen, on the other hand.     

Fractionation of plasma globulin for prothrombin,thrombokinase, and accessory thromboplastin

1. Crude globulin from more than 1,000 liters of citrated bovine plasma has been used in developing a procedure for moderately large scale separation of clotting factors. Fraction A, prothrombin, kinase, and thrombin fractions were prepared. Fraction A contained both kinase and accessory thromboplastin, the latter predominating when fraction A was diluted. 2. When prothrombin was activated by kinase, the rate of thrombin production was enhanced by the addition of platelets, or brain lipid, or dilute fraction A. These accessory thromboplastins caused this acceleration only when calcium chloride was added. Even with calcium, they were not effective unless kinase was present. 3. In contrast, the action of kinase was not entirely dependent on either ionic calcium or accessory thromboplastin. The concentrated kinase fraction activated prothrombin in the presence of excess oxalate. Although kinase often contaminates highly purified thrombins, it is probably distinct from thrombin. The ratio of kinase to thrombin was 100 times as great in the kinase fraction as in the thrombin fraction. 4. The kinase fraction, diluted 45,000-fold, to protein-nitrogen concentrations as low as 0.02 microgram per ml., accelerated the conversion of crude prokinase in three-stage tests. 5. The findings are consistent with the following concept of the basic enzymatic mechanism: See PDF for Structure It is now added that calcium and accessory thromboplastin exert their effects by impinging on the basic mechanism, in a chemically secondary or indirect manner.     

Changes in leukocyte count, blood glucose and coagulation system in calves injected with Escherichia coli endotoxin

Responses of leukocyte, blood glucose and coagulation system in calves were investigated to injection with Escherichia coli endotoxin. Severe leukopenia and hyperglycemia following transient hypoglycemia were noted within 24 hours after injection. In the coagulation system, a definite decrease in platelet count, prolongation of prothrombin time and activated partial thromboplastin time were observed. Fibrinogen, soluble fibrin monomer complex and clotting time, however, varied.     

玉米芯木聚糖硫酸酯抗凝血活性及其机制的研究

采用活化部分凝血活酶时间(APTT)、凝血酶时间(TT)和凝血酶原时间(PT)检测了玉米芯木聚糖硫酸酯(wisX-SB)的抗凝活性,结果表明wisX-SB能明显延长APTT和TT,而不影响PT,提示wisX-SB是通过内源性和/或共同途径发挥抗凝血作用的。采用发色底物法及纤维蛋白原转化实验分别考察了wisX-SB对凝血酶及纤维蛋白原的作用,结果提示wisX-SB的抗凝机制包括:直接抑制纤维蛋白原的转化;通过增强抗凝血酶III(AT-III)的活性,抑制凝血酶活性,从而达到抗凝目的。较低浓度时以前者为主,较高浓度下两者皆起作用。     

A simple,general approach of allosteric coagulation enzyme inhibition through monosulfated hydrophobic scaffolds

Allosteric inhibition of coagulation enzymes offers the advantage of controlled inhibition. In this study, a small library of mono sulfated indole and benzothiazole based molecules was synthesized and screened against the panel of coagulation proteases. The results reveal that selected molecules inhibit the thrombin, factor Xa and factor XIa with moderate potency. Compound 6a was found to have an allosteric mode of inhibition against thrombin. Plasma clotting assays suggest that selected inhibitors 14b, 14c and 14d prolong both prothrombin and activated partial thromboplastin time. Overall, this work presents the newer class of allosteric inhibitors of thrombin and factor XIa with improved aqueous solubility profile.     

Characterization of the fibrin polymer structure that accelerates thrombin cleavage of plasma factor XIII

The effect of plasmin-derived fibrin(ogen) degradation products on alpha-thrombin cleavage of plasma Factor XIII was studied to identify the fibrin polymer structure that promotes Factor XIIIa formation. Fibrin polymers derived from fibrinogen and Fragment X enhanced the rate of thrombin cleavage of plasma Factor XIII in plasma or buffered solutions. The concentrations of fibrinogen and Fragment X that promoted half-maximal rates of Factor XIIIa formation were 5 and 40 micrograms/ml, respectively. Fragments Y, D, E, D-dimer, and photooxidized fibrinogen did not enhance thrombin cleavage of Factor XIII. Although purified Fragment D1 inhibited fibrin gelation, the soluble protofibrils promoted thrombin activation of Factor XIII. Noncrosslinked fibrin fibers failed to enhance thrombin cleavage of Factor XIII. In conclusion, soluble fibrin oligomers function to promote thrombin cleavage of plasma Factor XIII during blood clotting.     

Effect of a High-Fat Diet on the Concentration of Antihemophilic Globulin and Factor V

Experiments were carried out to determine whether a diet known to produce coronary thrombosis in rats (Hartroft, O''Neal and Thomas⁶) could produce increases in antihemophilic globulin (AHG), Factor V, and other coagulation factors. In animals continuously fed the thrombogenic diet the concentration of AHG increased to 186% of normal by two weeks and to 373% by eight weeks. The concentration of Factor V rose to a lesser extent, reaching 138% compared to a normal value of 100%. The prothrombin time, stypven time and plasma clotting time also showed significant changes. After thrombin injections, the mortality from massive thrombosis was significantly higher in fat-fed rats. As a considerable number of rats on this diet were shown to develop spontaneous thrombosis, the coagulation disturbances here described acquire added importance.     

A Novel Factor Xa-Inhibiting Peptide from Centipedes Venom

Centipedes have been used as traditional medicine for thousands of years in China. Centipede venoms consist of many biochemical peptides and proteins. Factor Xa (FXa) is a serine endopeptidase that plays the key role in blood coagulation, and has been used as a new target for anti-thrombotic drug development. A novel FXa inhibitor, a natural peptide with the sequence of Thr-Asn-Gly-Tyr-Thr (TNGYT), was isolated from the venom of Scolopendra subspinipes mutilans using a combination of size-exclusion and reverse-phase chromatography. The molecular weight of the TNGYT peptide was 554.3 Da measured by electrospray ionization mass spectrometry. The amino acid sequence of TNGYT was determined by Edman degradation. TNGYT inhibited the activity of FXa in a dose-dependent manner with an IC₅₀ value of 41.14 mg/ml. It prolonged the partial thromboplastin time and prothrombin time in both in vitro and ex vivo assays. It also significantly prolonged whole blood clotting time and bleeding time in mice. This is the first report that an FXa inhibiting peptide was isolated from centipedes venom.     

Coagulopathy after snake bite by Bothrops neuwiedi: case report and results of in vitro experiments

Coagulation studies were performed in a patient who had been bitten by a snake of the species Bothrops neuwiedi. The patient presented with hemorrhagic necrosis at the envenomization site and considerable bleeding from venous puncture sites. He developed a severe defibrination syndrome with a clottable fibrinogen level of approximately 0.1 g/l. Fibrinogen was not measurable by clotting time assay. Fibrin degradation products were greatly elevated. Treatment with antivenom caused an anaphylactic reaction within ten minutes and serum sickness after three days. In vitro experiments revealed that B. neuwiedi venom directly activates Factors II and X, but does not activate Factor XIII. In vivo consumption of Factor XIII after B. neuwiedi envenomization is ascribed to the action of Factor IIa. At low venom concentrations clotting is initiated by activation of prothrombin by the venom either directly or via Factor X activation. Treatment with heparin might be beneficial in coagulopathy secondary to snake bite by reducing circulating active thrombin. The venom contains thrombin-like proteases which cause slow clotting of fibrinogen, and plasmin-like components causing further proteolysis of fibrinogen and fibrin. Antivenom has no effect on the proteolytic action of the snake venom. The in vivo effects of antivenom are presumably caused by acceleration of the elimination of venom components from the circulation. Intravenous administration of antivenom caused normalization of blood coagulation parameters within 48 h.     

Improved Procedures for the Purification of Selected Vitamin K-Dependent Proteins

Improved methods are described to obtain bovine prothrombin, Factor IX, Protein C, and autoprothrombin III (Factor X, Auto-III) in purified form. The prothrombin had a specific activity of 4, 340 Iowa units/mg. Theoretically, a preparation of clean thrombin should have a specific activity of 8, 200 U/mg, because 47.08% of the protein in prothrombin is lost when thrombin forms. Such thrombin preparations have been obtained (Arch. Biochem. Biophys. 121, 372 (1967)). The prothrombin concentration of bovine plasma is near 60 mg/liter. Protein C, first isolated by Stenflo (J. Biol. Chem. 251, 355 (1976)), was found to be the precursor of autoprothrombin II-A (Auto-II-A), discovered earlier (Thromb. Diath. Haemorrh. 5, 218 (1960)). Protein C (Factor XIV) was converted to Auto-II-A (Factor XlVa) by thrombin. Digesting purified Auto-III with purified thrombin removed a small glycopeptide from the COOH-terminal end of the heavy chain to yield Auto-IIItm. Auto-III throtnbin Auto-IIIm + peptide. Auto-IIIm was not converted to the active enzyme with thromboplastin and, furthermore, inhibited the activation of purified native Auto-III with thromboplastin. Auto-11 Im was also not converted to the active enzymewhen the procoagulants consisted of purified Factor VIII, purified Factor IXa, platelet factor 3 and calcium ions. The “activation peptide” released by RVV-X from the NH₂-terminal end of the heavy chain and the active enzyme (Auto-Cm) were purified. Auto-III was also activated with purified RVV-X. The same “activation peptide” was isolated, but Auto-C was obtained instead of Auto-Cm. Purified Factor IX developed anticoagulant activity when reacted with an optimum concentration of purified thrombin. A suitable reagent for the assay of Factor IX was prepared by removing prothrombin complex from anticoagulated bovine plasma and restoring the prothrombin and Auto-III concentration with use of the respective purified proenzymes.     

Effect of polyanetholesulphonic acid and xylan sulphate on antithrombin III activity.

Both polyanetholesulphonic acid and xylan sulphate prolonged the partial thromboplastin clotting time of plasma. The anticoagulant effect of both compounds was reduced following pre-incubation of plasma with antiserum specific for antithrombin III. Polyanetholesulphonic acid was more effective than xylan sulphate in inhibiting thrombin-initiated clotting of plasma, and potentiated antithrombin III inhibition of both thrombin and Xa. Xylan sulphate was more effective in potentiating antithrombin III inhibition of Xa than of thrombin. These differential effects of xylan sulphate on different blood serine proteases are discussed in terms of the antithrombin III-mediated anticoagulant activity of heparin.     

Hypocoagulable state of human preovulatory ovarian follicular fluid: role of sulfated proteoglycan and tissue factor pathway inhibitor in the fluid

Ovulation accompanied by tissue damage can cause an increase in the level of tissue factor (TF) in the follicular fluid, triggering the extrinsic coagulation pathway. However, follicular fluid must block fibrin formation and maintain fluidity until the release of the oocyte at ovulation. The combination of sulfated proteoglycan, antithrombin, and TF pathway inhibitor (TFPI) appears to play a critical role in the hypocoagulability of human follicular fluid. When compared with plasma, folicular fluid differs markedly in the levels of a number of important coagulation proteins. Principal among these are 15-fold, 13-fold, and 3.7-fold increases in free TFPI, thrombin-antithrombin complex, and TF, respectively. The excessively prolonged activated partial thromboplastin time (APTT) and prothrombin time (PT) of human ovarian follicular fluid appear to be primarily due to high concentrations of sulfated proteoglycans, which accelerate the inactivation of thrombin and the anti-Xa activity of TFPI. Thus, heparitinase treatment shortened the clotting times of follicular fluid and reduced the inhibition of thrombin by the proteoglycan fraction combined with a fraction containing antithrombin. The remaining prolongation of APTT and PT may be caused by high levels of free TFPI in follicular fluid, which were confirmed by Northern blotting analysis, demonstrating TFPI mRNA expression by granulosa cells.