The influence of the surface charge of phospholipids on the activity of the prothrombin converting complex

During blood coagulation factor V is converted into its activated form, factor Va, by the action of thrombin. In the presence of Ca⁺⁺-ions, phospholipids, factor Xa and factor Va, a complex is formed, prothrombinase, which converts prothrombin into thrombin. The influence of the surface charge of various phospholipid micelles on the formation of this complex was studied. As phospholipids purified phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositin (PI), phosphatidylcholine (PC) and mixed micelles of some of these phospholipids were used.

The experiments revealed that after the addition of phospholipids to factors Va, Xa, II and Ca⁺⁺-ions two reaction phases could be distinguished. During the first phase a fall of factor Va activity occurred, which was faster as the negative charge of the micelles increased. The presence of factors II and Xa was not required for this reaction. This fall in activity suggests that Ca⁺⁺-ions are needed for the stability of factor Va. During the second phase maximal reduction rate of factor Va activity, accompanied by maximal thrombin generation from prothrombin, occurred only in the case of an optimal negative surface charge of the phospholipid micelles. The presence of factors II and Xa was essential for this reaction. The second reaction phase probably implies consumption of factor Va in favour of thrombin generation.     

Anticoagulant activity of beta 2-glycoprotein I is potentiated by a distinct subgroup of anticardiolipin antibodies.

Plasmas of 16 patients positive for both IgG anticardiolipin (aCL) antibodies and lupus anticoagulant (LA) antibodies were subjected to adsorption with liposomes containing cardiolipin. In 5 of these plasmas both the anticardiolipin and the anticoagulant activities were co-sedimented with the liposomes in a dose-dependent manner, whereas in the remaining cases only the anticardiolipin activity could be removed by the liposomes, leaving the anticoagulant activity (LA) in the supernatant plasma. aCL antibodies purified from the first 5 plasmas were defined as aCL-type A, while the term aCL-type B was used for antibodies in the other 11 plasmas, from which 2 were selected for this study. Prolongation of the dRVVT was produced by affinity-purified aCL-type A antibodies in plasma of human as well as animal (bovine, rat and goat) origin. aCL-type B antibodies were found to be devoid of anticoagulant activity, while the corresponding supernatants containing LA IgG produced prolongation of the dRVVT only in human plasma. These anticoagulant activities of aCL-type A and of LA IgG's were subsequently evaluated in human plasma depleted of beta 2-glycoprotein I (beta 2-GPI), a protein which was previously shown to be essential in the binding of aCL antibodies to anionic phospholipids. Prolongation of the dRVVT by aCL-type A antibodies was abolished using beta 2-GPI deficient plasma, but could be restored upon addition of beta 2-GPI. In contrast, LA IgG caused prolongation of the dRVVT irrespective of the presence or absence of beta 2-GPI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Importance of N-terminal residues in plasminogen activator inhibitor 1 on its antibody induced latency transition

Bothrojaracin (BJC) is a 27 kDa snake venom protein from Bothrops jararaca that has been characterized as a potent ligand (KD = 75 nM) of human prothrombin (Monteiro RQ, Bock PE, Bianconi ML, Zingali RB, Protein Sci 2001; 10: 1897-904). BJC binds to the partially exposed anion-binding exosite I (proexosite I) forming a stable 1:1, non-covalent complex with the zymogen whereas no interaction with fragment 1 or 2 domains is observed. In addition, BJC interacts with thrombin through exosites I and II (KD = 0.7 nM), and influences but does not block the proteinase catalytic site. In the present work we studied the effect of BJC on human prothrombin activation by factor Xa in the absence or in the presence of its cofactors, factor Va and phospholipids. In the absence of phospholipids, BJC strongly inhibited (80%) the zymogen activation by factor Xa in the presence but not in the absence of factor Va, suggesting a specific interference in the cofactor activity. In the presence of phospholipid vesicles (75% phosphatidylcholine, 25% phosphatidylserine), BJC also inhibited (35%) prothrombin activation by factor Xa in the presence but not in the absence of factor Va. BJC showed a higher inhibitory effect (70%) towards thrombin formation by prothrombinase complex assembled on phospholipid vesicles composed by 95% phosphatidylcholine, 5% phosphatidylserine. Activation of prothrombin by platelet-assembled prothrombinase complex (factor Xa, factor Va and thrombin-activated platelets) showed that hirudin (SO3-) and BJC efficiently inhibit the thrombin formation (43% and 84%, respectively). Taken together, our results suggest that proexosite I blockage decreases the productive recognition of prothrombin as substrate by factor Xa-factor Va complex and prothrombinase complex. Furthermore, data obtained with human platelets suggest that proexosite I may play an important role in the physiological activation of prothrombin.     

Pseutarin C,a prothrombin activator from Pseudonaja textilis venom: its structural and functional similarity to mammalian coagulation factor Xa-Va complex

Several snake venoms contain procoagulant proteins that can activate prothrombin. We have purified pseutarin C, a prothrombin activator from the venom of the Australian brown snake (Pseudonaja textilis). It converts prothrombin to thrombin by cleaving both the peptide bonds Arg(274)-Thr(275) and Arg(323)-Ile(324), similar to mammalian factor Xa. It is a protein complex (approximately 250 Kd) consisting of an enzymatic and a non- enzymatic subunit. These subunits were separated by reverse phase HPLC and their interactions with bovine factor Xa and factor Va were studied. The enzymatic subunit of pseutarin C has an approximately 13 fold higher affinity for bovine factor Va (K(d) of 11.4 nM for pseutarin C enzymatic subunit--bovine factor Va interaction as compared to a K(d) of 147.4 nM for the bovine factor Xa-Va interaction). The non-enzymatic component, however, was unable to activate bovine factor Xa. N-terminal sequence analysis of the catalytic subunit of pseutarin C showed approximately 60% homology to mammalian factor Xa and approximately 78% homology to trocarin, a group D prothrombin activator from Tropidechis carinatus venom. Structural information for the non-enzymatic subunit of pseutarin C was obtained by amino terminal sequencing of several internal peptides. The sequence data obtained indicates that the non-enzymatic subunit of pseutarin C has similar domain architecture like the mammalian factor Va and the overall homology is approximately 55%. Thus pseutarin C is the first venom procoagulant protein that is structurally and functionally similar to mammalian factor Xa-Va complex.     

The effects of bovine prothrombin fragment 1 and fragment 1.2 on prothrombin activation

In this paper we describe the effects of the activation peptides prothrombin fragment 1 and fragment 1.2 on factor Xa-catalyzed prothrombin activation. Prothrombin activation in free solution by either factor Xa or factor Xa together with factor Va is unaffected by the activation fragments. When negatively charged phospholipids are present we observed considerable inhibition of prothrombin activation by both fragment 1 and fragment 1.2. For the activation of 0.25 microM prothrombin by factor Xa in the presence of 50 microM phospholipid (phosphatidylserine/phosphatidylcholine, 25/75; mol/mol) and 5 mM CaCl2 50% inhibition was obtained at 0.28 microM fragment 1 or fragment 1.2. Much higher fragment concentrations were required for 50% inhibition of a prothrombinase complex consisting of factor Xa, factor Va, Ca2+ and phospholipid. This shows that factor Va protects prothrombin activation against inhibition by its own activation peptides. Less inhibition by activation fragments was also observed at higher phospholipid and prothrombin concentrations or when the mole fraction phosphatidylserine in the phospholipid vesicles was decreased. The effects of fragment 1 and fragment 1.2 on prothrombin activation were identical throughout all experiments, indicating that the inhibition is due to the gamma-carboxyglutamic acid containing region of the activation peptides. Our observations suggest that the activation fragments inhibit prothrombin activation by competing with prothrombin and factor Xa for binding sites at the phospholipid surface. In such a model factor Va will protect against the inhibition since it is known to promote the assembly of the prothrombinase complex through interactions with factor Xa and prothrombin that are independent of the gla-residues. The kinetic properties of fragment inhibition also suggest that in vivo prothrombin activation will not be affected by the generation of activation peptides.     

Two separable steps in the development of platelet prothrombin-converting activity

Platelets catalyze conversion of prothrombin to thrombin in the presence of coagulation Factors Va and Xa. Bovine Factor Va was found to bind to human and bovine platelets washed by centrifugal pelleting. Factor Va was bound to sites varying in affinities but only those sites saturated at approximately 1 mM are active. Platelets in citrate or benzamidine-anticoagulated plasma or those washed by gel filtration bound substantially less Factor Va with these affinities, indicating that the binding sites increased in number or affinity or both during the centrifugal pelleting and redispersal. Thrombin production catalyzed by platelets with developed binding sites started at a low initial rate but after a lag period rapidly reached a maximum rate. Addition of 0.25 mM dinitrophenol increased the lag period and decreased the maximum rate obtained concomitantly with the reduction in ATP levels. Neither dinitrophenol nor its induced depletion of the ATP levels reduced Factor Va binding. Therefore, the activation of platelet prothrombin-converting activity was separated into two steps; one being the development of the high-affinity binding sites and the other a step partially dependent on sufficient levels of ATP within the platelet.     

A peptide derived from human prothrombin fragment 2 inhibits prothrombinase and angiogenesis

We constructed the synthetic peptide library representing human prothrombin fragment 2 (F2) sequence and explored the inhibitory sequence for prothrombinase, which was reconstituted in vitro by adding factor Xa, factor Va, and calcium into phospholipids. The nonapeptide NSAVLQVEN (NSA9) suppressed prothrombinase reconstituted not only on phospholipid vesicles but also on the bovine capillary endothelial (BCE) cell surface. Kinetic analyses demonstrated that NSA9 is a mixed-type inhibitor of Xa. Furthermore, the nonapeptide inhibited the proliferation of BCE cells and also suppressed angiogenesis in chicken embryos. The inhibitory activities of NSA9 were abrogated by pre-incubation with anti-F2 monoclonal antibody, 4E7. These data demonstrate that anti-angiogenic activity of F2 may be related to its ability to inhibit prothrombinase.     

Regulation of prothrombinase activity by protein S.

The independent effect of protein S as prothrombinase inhibitor has been proposed to depend on binding to both coagulation factors Va and factor Xa or on the binding to phospholipid thereby limiting the phospholipid available for prothrombinase activity. In this study we show that plasma concentrations of protein S (300 nM) equilibrated with the prothrombinase components (factor Va, factor Xa, phospholipid) cause a profound inhibition at low phospholipid concentrations (approximately 0.2 microM). This inhibition by protein S of prothrombinase activity is abrogated with increasing phospholipid concentrations. Modeling of the effect of protein S on prothrombinase based only on the reported affinity of protein S for phospholipids (Kd approximately 10(-8) M) in an equilibrium model (Clotspeed), predicted the experimentally obtained thrombin generation rates at low phospholipid in the presence of protein S based on the diminished available phospholipid binding sites for the prothrombinase components. Consistently, initial rates of prothrombinase activity are already maximally inhibited when protein S is preincubated with the phospholipid prior to the addition of factor Xa, factor Va and prothrombin. The results indicate that the order of addition of prothrombinase components and the availability of phospholipid may have a profound influence on observed effects of protein S on prothrombinase activity. All prothrombinase components (factor Xa, factor Va, phospholipid) become available during the course of the physiological thrombin generation. The effect of protein S was therefore studied on tissue factor-induced, platelet-dependent thrombin generation. Protein S delayed and inhibited the rate of thrombin generation of tissue factor-induced thrombin formation when surface is provided at physiologic concentrations using isolated platelets (2 x 10(8)/ml). In contrast, protein S hardly affected thrombin generation in this model when platelets were pre-activated with collagen. Furthermore, the observed effects of addition of protein C and thrombomodulin in the absence or presence of protein S on tissue factor-induced, platelet-dependent thrombin generation, indicate that protein S and protein C may cooperate in the regulation of prothrombinase activity through independent mechanisms.     

Immunological aspects of some vitamin K-dependent factors and preparation of depleted plasmas

The specificity of the immune response elicited in rabbits to purified bovine prothrombin, autoprothrombin III (Factor X), Factor IX, and Protein C was studied. The immunizing proteins were homogeneous on polyacrylamide gel electrophoresis. By immunodiffusion analysis antigenic similarities were not detected between them in spite of known stretches of amino acid sequence homology. Immunoelectrophoresis of bovine plasma and each purified protein gave identical single precipitin lines with the homologous antisera. This experiment established that the antibodies prepared against each one of the purified vitamin K-dependent factors studied reacted with a single component in bovine plasma. Immunologic crossreactions were found between human, bovine, dog, rat and chicken prothrombin, Protein C, Factor X, and Factor IX. They were not species specific. Purified bovine prothrombin has at least four antigenic determinant sites. Incubation of the purified proteins with suitable proteolytic enzymes yielded fragments and intermediate products which were purified and tested for their potential capacity to react with antibodies in the native antisera. In immunoelectrophoresis tests both prothrombin fragments 1 and 2 and the intermediate product prethrombin 1 gave immunoprecipitates which were separately identified by their differing mobilities, while the enzyme portion, thrombin, did not react with antithrombin serum. In addition to prothrombin, bovine plasma contained prethrombin 1, prothrombin fragments 1 and 2, as well as prethrombin 2 and/or thrombin. Autoprothrombin III$\text{m}$ (Factor Xβ) and autoprothrombin C (Factor Xa) crossreacted with anti-autoprothrombin III and the active form of Protein C. Gamma globulins of antisera to prothrombin, to autoprothrombin III, to Factor IX, and to Protein C were fractionated by using saturation ammonium sulfate solution and coupled to activated Agarose A-15m beads. The insolubilized antibodies were used to absorb the antigen from the plasma. Each depleted plasma served as test material for a specific clotting factor. The prolonged prothrombin time of the prothrombin depleted plasma was normalized by additions of purified prothrombin, but not by purified prethrombin 1. The prothrombin time of autoprothrombin III depleted plasma was shortened by fresh plasma, purified autoprothrombin III, and by purified autoprothrombin III$\text{m}$ (Factor Xβ). In the case of Factor IX depleted plasma, the partial thromboplastin time was used. The depleted bovine plasmas are suitable for assay purposes. They serve the same role as the corresponding deficient plasma in bioassay coagulation tests.     

Mechanisms of α-thrombin and β-thrombin-E formation: Use of ecarin for isolation of meizothrombin 1

Purified Ecarin from $\text{Echis carinatus}$ venom converted purified bovine prethrombin 1 to meizothrombin 1. This involved only the breaking of an Arg-Ile bond. Meizothrombin 1 was purified. It was free of α-thrombin and was a single component by polyacrylamide gel electrophoresis. It had 14.5 times more esterase activity than proteolytic activity on fibrinogen. Like α-thrombin it degraded purified prothrombin to prethrombin 1. Meizothrombin 1 itself converted to α-thrombin plus prothrombin fragment 2 by autolysis, and also with purified Factor Xa. In the latter case, Factor V plus phospholipids or sodium taurocholate plus calcium ions accelerated the reaction. The amount of Factor Xa required in the presence of accessories was far less than needed for prothrombin as the substrate. The α-thrombin which formed converted to β-thrombin-E by autolysis. Purified meizothrombin 1 was dried from the frozen state without loss of activity, and the activity was stable at ?20°C in 50% glycerol solution containing 0.05M phosphate buffer pH 6.5 and benzamidine 0.001M. A likely sequence for thrombin formation by Factor Xa begins with the production of meizothrombin possessing strong esterase and weak coagulant activity. Next, meizothrombin is converted to α-thrombin plus prothrombin fragment 1.2.     

Collagen derived octapeptide inhibits platelet procoagulant activity induced by the combined action of collagen and thrombin

Platelet prothrombin converting activity was measured in a system using washed human platelets and purified coagulation factors Xa, Va and prothrombin. Exposure of platelet prothrombin converting activity evoked by collagen or the combined action of collagen and thrombin was effectively inhibited when a collagen derived octapeptide was added prior to platelet activation. Half maximal inhibition of prothrombinase activity of platelets stimulated by collagen plus thrombin- or collagen alone was obtained at 0.9 mM and 0.5 mM octapeptide, respectively. This suggests a modifying effect of thrombin on the platelet-collagen interaction. Octapeptide either alone or in combination with thrombin was unable to enhance platelet procoagulant activity. The increased prothrombin converting activity seen upon treatment of platelets with ionophore A23187 was not affected by octapeptide, added either before or after treatment with ionophore. It is concluded that octapeptide specifically interferes with the platelet-collagen interaction required to generate a procoagulant surface which enhances the rate of thrombin formation.     

Prothrombin Clamart: prothrombin variant with defective Arg 320-IIe cleavage by factor Xa

An abnormal prothrombin has been detected in a 23 yr-old healthy female and her mother. Both patients appeared to be heterozygous for the abnormality, plasma prothrombin being 50% of normal using the usual one stage assay, but normal when measured either by using Echis carinatus venom or by immunoassay. No abnormality in the immunoelectrophoretic pattern was observed. Prothrombin isolation on DEAE Sephadex failed to separate the abnormal population (prothrombin Clamart) from the normal one. The rates of prothrombin activation by factor Xa, in the presence or absence of phospholipids and/or factor Va, were determined by measuring the production of both clotting and amidolytic activities. The thrombin generation rate from prothrombin isolated from the propositus plasma was 50% slower than normal whatever the method of measurement and the composition of the activation mixture. Analysis of the final activation products by SDS polyacrylamide gel electrophoresis revealed that equal amounts of prethrombin 2 and thrombin had been formed. Prethrombin 2 Clamart was shown to be resistant to proteolysis upon further incubation with factor Xa, whereas it was readily converted to thrombin by Echis carinatus venom. Prothrombin Clamart appears to be characterized by an impairment of Arg 320-IIe cleavage by factor Xa.     

Localization of the inhibitory site(s) of pentosan polysulphate in blood coagulation

We studied the inhibitory effect of pentosan polysulphate (PPS, Hémoclar) on thrombin formation in blood coagulation. In contrast to a current hypothesis the antithrombin III independent effect of PPS on blood coagulation is not caused by preventing the binding of the factors IX, IXa, X, Xa, VIII, V, Va and II onto procoagulant phospholipids. We investigated the activation by thrombin of factors I, V and VIII. A strong inhibitory effect of PPS on factor VIII activation could be observed. Inhibition of the activation of factor V to the same extent requires about 30-fold higher concentrations of PPS, whereas the activation (clotting) of fibrinogen is not inhibited. The effect of PPS on factor VIIIa is two-fold: A) it inhibits its formation and B) it inhibits its function probably by the formation of a factor VIIIa-PPS complex. Prothrombinase, constituted of purified factors Xa, Va and phospholipids was not inhibited by PPS, neither were incomplete forms of this enzyme, lacking phospholipids or factor Va. The complete factor X activating enzyme (factors IXa, VIIIa and phospholipids), however, was strongly inhibited, but incomplete forms, lacking factor VIII, were not. The inhibition of the complete enzyme can be explained by reversible binding of PPS to factor VIIIa (causing an inhibition of its function) and it is not an effect on the enzymatic function of the complete enzyme. On saturation of the enzyme with an excess of factor VIIIa no inhibition by PPS is noticed. We postulate therefore that the antithrombin III independent inhibitory effect of PPS on thrombin generation on blood coagulation is by interaction with factor VIIIa. This effect is additional to the heparin-like action of PPS, i.e. potentiation of the activity of antithrombin III and/or heparin cofactor II.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prothrombin activation in rabbits

The suitability of rabbit prothrombin activation fragment F 1.2 as a marker for the activation of the coagulation system was tested. Monoclonal antibodies to rabbit F 1.2 were raised, and a competitive F 1.2 ELISA was developed. Within the detection limit of the ELISA, no increase in rabbit F 1.2 was detected upon recalcification of plasma, whereas human F 1.2 increased 1500-fold. The apparent lack of F 1.2 formation in rabbit serum was confirmed by immunoblotting analysis of endogenous and biotin-labeled prothrombin. Meizothrombin and the B-chain of thrombin were the only prothrombin fragments detectable. In contrast, labeled human prothrombin formed, in addition, prethrombin 2 and F 1.2 in both human and rabbit serum. In contrast, rabbit F 1.2 formation could be demonstrated using purified rabbit prothrombin and factor Xa. These observations raise the possibility that rabbit prothrombin is less susceptible than the human counterpart to factor Xa cleavage at the 271/272 peptide bond. Thus, the primary structure of rabbit prothrombin was deduced by cDNA sequencing. While the 320/321 Xa cleavage site giving rise to meizothrombin was identical in rabbit and human prothrombin, the flanking region of the 271/272 Xa sensitive site contained a six amino acid deletion in the rabbit sequence. Taken together, these observations suggest that the observed differences between human and rabbit prothrombin activation may be due to different susceptibilities of the two Xa cleavage sites rather than plasma or serum cofactor(s).     

Phospholipid requirements in thrombin formation

Lipids normally found in plasma do not appreciably effect formation of thrombin from prothrombin. This was demonstrated by finding that normal and lipid-poor plasma clotted almost with equal rapidity when calcium ions and purified autoprothrombin C (Factor Xa) were added. Lipid-poor plasma and normal plasma served equally well to test the lipid requirements of autoprothrombin C in thrombin formation. Of the purified phospholipids tested, phosphatidylserine and phosphatidylinositol were the most effective. Whole brain thromboplastin or its lipid portion alone or platelet factor 3 preparations also accelerated thrombin formation. Phosphatidylethanolamine and choline were virtually ineffective. The lipid requirements for autoprothrombin C formation are different from those required by autoprothrombin C to generate thrombin from prothrombin.     

Mechanism of anticoagulant action by protein inhibitors from bovine testes and salmon sperm

A protein inhibitor was isolated from commercial preparations of salmon sperm and its physical and anticoagulant properties were compared with an inhibitor isolated earlier from commercial preparation of bovine testicular hyaluronidase. The inhibitor from bovine source was heat and acid labile and had a molecular weight of approximately equal to 35000 while the one from salmon sperm had a molecular weight of approximately equal to 5700 and was stable to heat and acid. To determine the mechanism of the inhibitory effect, a system of purified components consisting of isolated prothrombin, Factor Xa, Factor Va, Ca++, and vesicles of phosphatidylcholine (PCPS, 25% PS) was used. Included also was dansylarginine N- (3-ethyl-1,5-pentanedidyl) amide (DAPA) which binds newly formed thrombin and yields the time course of prothrombin conversion by virtue of enhanced fluorescence of the DAPA - thrombin complex. The inhibitor of bovine testes was effective only when PCPS was the limiting component suggesting that its action was directed against the phospholipid component of the prothrombinase complex. The inhibitor from salmon sperm was found to lower the rate of conversion of prothrombin to thrombin in an in vitro system where thrombin generation was measured by its action on the chromogenic substrate H-D-Phe-Pip-Arg-pNa (S-2238). It inhibited the conversion of Factor X to Xa and also the the amidolytic cleavage by Factor Xa of chromogenic substrate N-Benz-Ile-Glu-Gly-Arg-pNa (S-2222).     

Clinical significance of various ELISA assays for detecting antiphospholipid antibodies

We have compared the prevalence of antiphospholipid antibodies (APA) measured by enzyme-linked immunosorbent assay (ELISA), in 119 selected patients using five different antigens: bovine cardiolipin, phosphatidylserine, phosphatidylinositol, bovine partial thromboplastin and human brain partial thromboplastin. All the plasmas have been evaluated for the presence of lupus anticoagulant (LA) activity by clotting techniques. We found a significant association between the incidence of LA and APA (p less than 0.001), only moderate agreement between the prolongation of the activated partial thromboplastin time (APTT) and ELISAs (r around 0.50) and a good agreement between the ELISAs (r around 0.80). The combination of antibodies against cardiolipin (ACA) and human brain partial thromboplastin (AHPTA) allowed the detection of antibodies in most of the LA positive cases. ACA, AHPTA and antiphosphatidylinositol antibodies detected all the positive samples. Six patients (5%) had a single APA detected. The clinical associations of APA according to phospholipid specificity, immunoglobulin isotype and titer are shown.     

Factor IX activity derived from purified prothrombin

Factor IX-like activity could be generated from highly purified prothrombin in the presence of phospholipids and CaCl₂ when BaCO₃-adsorbed Factor XI deficient plasma and Factor XIa were present, when BaSO₄-adsorbed hemophilia B plasma was present or when only Factor XIa was present. Factor IX-like activity was observed to increase when prothrombin, Factor XIa, phospholipids and CaCl₂ were incubated while prothrombin activity was observed to disappear. A 3-hour incubation sample was shown to correct only hemophilia B plasma; it did not correct hemophilia A nor Factor X deficient plasmas.     

Regulation of bovine activated protein C by protein S: the role of the cofactor protein in species specificity

The anticoagulant activity of bovine activated Protein C was observed to exhibit species specificity when tested in the plasmas from cow, dog, rabbit, and human. The relative effectiveness in descending order was cow, dog, human, and rabbit. When bovine Protein S was added to each of these plasmas, the species specificity of bovine activated Protein C was lost. No activated Protein C cofactor activity could be detected in either rabbit or human plasma. Bovine activated Protein C enhanced the rate of inactivation of both rabbit and human Factor Va in serum. The rate of activated Protein C catalyzed inactivation of rabbit and human Factor Va could be enhanced by the addition of bovine Protein S. These results indicated that the species specificity of the anticoagulant activity of bovine activated Protein C is mediated by a cofactorenzyme interaction rather than an enzyme-substrate interaction. These results further demonstrated that the anticoagulant activity of activated Protein C is due, in part, to the inactivation of Factor Va.     

Continuous flow and the prothrombinase-catalyzed activation of prothrombin

The activation of prothrombin by prothrombinase was investigated in a continuous flow system at 25 degrees C. A glass capillary, containing a continuous phospholipid bilayer attached to the interior surface, was first perfused with factor Va. The factor Va bound to the phospholipid surface functioned as sites for the formation of prothrombinase, when subsequently a factor Xa and prothrombin containing solution was perfused. Under the conditions used, steady-state rates of prothrombin activation were attained after 4 to 15 min. The rates of prothrombinase formation increased with increasing factor Xa concentrations and flow rates, which is compatible with the assembly of prothrombinase being dependent on the flux of factor Xa to the phospholipid-bound factor Va. As long as factor Xa and prothrombin were present in the fluid phase the assembly of prothrombinase was apparently irreversible; during at least 20 min no loss of activity occurred. The steady-state rate of prothrombin activation was dependent on the surface concentration of prothrombinase, at 1.0 microM prothrombin and a shear rate of 82 s-1 the average rate was 870 mol thrombin/min per mol prothrombinase. In contrast to test tube experiments it was observed that in this flow system, the formation of alpha-thrombin is favoured above the formation of meizothrombin (des fragment 1).