Table grape consumption reduces adiposity and markers of hepatic lipogenesis and alters gut microbiota in butter fat-fed mice

Our objective was to determine if consuming table grapes reduces adiposity and its metabolic consequences and alters gut microbiota in mice fed a high-fat (HF), butter-rich diet. C57BL/6 J mice were fed a low-fat (LF) diet or HF diet with 3% or 5% grapes for 11 weeks. Total body and inguinal fat were moderately but significantly reduced in mice fed both levels of grapes compared to their controls. Mice fed 5% grapes had lower liver weights and triglyceride levels and decreased expression of glycerol-3-phosphate acyltransferase (Gpat1) compared to the 5% controls. Mice fed 3% grapes had lower hepatic mRNA levels of peroxisome proliferator-activated receptor gamma 2, sterol-CoA desaturase 1, fatty-acid binding protein 4 and Gpat1 compared to the 3% controls. Although grape feeding had only a minor impact on markers of inflammation or lipogenesis in adipose tissue or intestine, 3% of grapes decreased the intestinal abundance of sulfidogenic Desulfobacter spp. and the Bilophila wadsworthia-specific dissimilatory sulfite reductase gene and tended to increase the abundance of the beneficial bacterium Akkermansia muciniphila compared to controls. In addition, Bifidobacterium, Lactobacillus, Allobaculum and several other genera correlated negatively with adiposity. Allobaculum in particular was increased in the LF and 3% grapes groups compared to the HF-fed controls. Notably, grape feeding attenuated the HF-induced impairment in epithelial localization of the intestinal tight junction protein zonula occludens. Collectively, these data indicate that some of the adverse health consequences of consuming an HF diet rich in saturated fat can be attenuated by table grape consumption.     

Purification and characterization of the anticoagulant principle of Trimeresurus mucrosqualatus venom

By means of CM-Sephadex column chromatography, Trimeresurus mucrosquamatus venom was separated into 20 fractions. Fraction XX had the marked anticoagulant action. This fraction was refractionated three times on Sephadex G-75, and a single peak was obtained. The patterns of microzone and disc electrophoresis also showed a single band. A single, symmetrical boundary with a value of 1.61 S was obtained by ultracentrifugation. It was a single peptide chain with a molecular weight of 11 700. The isoelectric point was higher than pH 10.The anticoagulant principle possesses phospholipase A activity and was calcium ion dependent. It did not possess proteolytic, tosyl-L-arginine methyl ester esterase, phosphodiesterase and alkaline phosphomonoesterase activities of the crude venom. The phospholipase A activity was heat-labile at pH 7.4, but was heat-stable at pH 5.6. The anticoagulant activity was more resistant to heat treatment as compared with phospholipase A activity.The anticoagulant action of the purified principle was competitively inhibited by platelet phospholipid, tissue thromboplastin and cephalin, and was neutralized by antiserum. The anticoaugulant principle inhibited platelet aggregation induced by ADP. It did not destroy fibrinogen, Factor X, prothrombin and thrombin; nor did it induce fibrinolysis nor interfere with the interaction between thrombin and fibrinogen. It is concluded that the anticoagulant action of this phospholipase A was due to the inhibition of the activations of Factors X and II through the activation of the procoagulant activity of phospholipids mediated partly by phospholipid-binding activity of this venom enzyme and partly by its enzymatic hydrolysis of phospholipids.     

Follow-Up of Thrombin Generation after Prostate Cancer Surgery: Global Test for Increased Hypercoagulability

Recent studies provided evidence that evaluation of thrombin generation identifies patients at thrombotic risk. Thrombin generation has a central role in hemorrhage control and vascular occlusion and its measurement provides new metrics of these processes providing sufficient evaluation of an individual’s hemostatic competence and response to anticoagulant therapy. The objective of the study is to assess a new measure of hypercoagulability that predisposes to venous thromboembolism in the postoperative period after radical prostatectomy. Pre- (day-1) and postoperative (hour 1, day 6, month 1 and 10) blood samples of 24 patients were tested for plasma thrombin generation (peak thrombin), routine hematology and hemostasis. Patients received low molecular weight heparin for thromboprophylaxis. Peak thrombin levels were higher in patients compared to controls at baseline (p<0.001), and elevated further in the early postoperative period (p<0.001). Longer general anesthesia and high body mass index were associated with increased thrombin generation after surgery (p = 0.024 and p = 0.040). D dimer and fibrinogen levels were higher after radical prostatectomy (p = 0.001 and p<0.001). Conventional clotting tests remained within the reference range. Our study contributed to the cognition of the hypercoagulable state in cancer patients undergoing pelvic surgery and revealed the course of thrombin generation after radical prostatectomy. Whilst it is unsurprising that thrombin generation increases after tissue trauma, further evaluation of this condition during the postoperative period would lead urologists to an international and well-supported consensus regarding thromboprophylaxis in order to provide better clinical outcome. Considering the routine evaluation of procoagulant activity and extending prophylactic anticoagulant therapy accordingly may potentially prevent late thrombotic events.     

The thrombin mutant W215A/E217A shows safe and potent anticoagulant and antithrombotic effects in vivo

Administration of the thrombin mutant W215A/E217A (WE), rationally designed for selective activation of the anticoagulant protein C, elicits safe and potent anticoagulant and antithrombotic effects in a baboon model of platelet-dependent thrombosis. The lowest dose of WE tested (0.011 mg/kg bolus) reduced platelet thrombus accumulation by 80% and was at least as effective as the direct administration of 40-fold more (0.45 mg/kg bolus) activated protein C. WE-treated animals showed no detectable hemorrhage or organ failure. No procoagulant activity could be detected for up to 1 week in baboon plasma obtained following WE administration. These results show that engineered thrombin derivatives that selectively activate protein C may represent useful therapeutic agents for the treatment of thrombotic disorders.     

Issue information

Thrombosis is a leading cause of morbidity and mortality throughout the world. Thrombolytic agents are important for both the prevention and treatment of thrombosis. Fibrin clot and turbidity assays revealed that it was able to inhibit the formation of fibrin clot. Chlorogenic acid degraded blood clot and inhibited the enzymatic activity of procoagulant proteases, thrombin, activated factor X (FXa), and activated factor XIII (FXIIIa). Chlorogenic acid was found to delay activated partial thromboplastin time, prothrombin time, and thrombin time. PFA‐100 assays showed that it prolonged the closure time of citrated whole human blood. It demonstrated the antithrombotic effect in collagen and epinephrine‐induced acute thromboembolism mice model. These antithrombotic profiles together with its anticoagulant and platelet disaggregation properties, and lack of toxicity to NIH‐3T3 and 3T3‐L1 cells, make it a potential agent for thrombotic treatment and prevention.     

Probing the hirudin-thrombin interaction by incorporation of noncoded amino acids and molecular dynamics simulation

Thrombin is a primary target for the development of novel anticoagulants, since it plays two important and opposite roles in hemostasis: procoagulant and anticoagulant. All thrombin functions are influenced by Na+ binding, which triggers the transition of this enzyme from an anticoagulant (slow) form to a procoagulant (fast) form. In previous studies, we have conveniently produced by chemical synthesis analogues of the N-terminal fragment 1-47 of hirudin HM2 containing noncoded amino acids and displaying up to approximately 2700-fold more potent antithrombin activity, comparable to that of full-length hirudin. In the work presented here, we have exploited the versatility of chemical synthesis to probe the structural and energetic properties of the S3 site of thrombin through perturbations introduced in the structure of hirudin fragment 1-47. In particular, we have investigated the effects of systematic replacement of Tyr3 with noncoded amino acids retaining the aromatic nucleus of Tyr, as well as similar hydrophobic and steric properties, but possessing different electronic (e.g., p-fluoro-, p-iodo-, or p-nitro-Phe), charge (p-aminomethyl-Phe), or conformational (homo-Phe) properties. Our results indicate that the affinity of fragment 1-47 for thrombin is proportional to the desolvation free energy change upon complex formation, and is inversely related to the electric dipole moment of the amino acid side chain at position 3 of hirudin. In this study, we have also identified the key features that are responsible for the preferential binding of hirudin to the procoagulant (fast) form of thrombin. Strikingly, shaving at position 3, by Tyr --> Ala exchange, abolishes the differences in the affinity for thrombin allosteric forms, whereas a bulkier side chain (e.g., beta-naphthylalanine) improves binding preferentially to the fast form. These results provide strong, albeit indirect, evidence that the procoagulant (fast) form of thrombin is in a more open and accessible conformation with respect to the less forgiving structure it acquires in the slow form. This view is also supported by the results of molecular dynamics simulations conducted for 18 ns on free thrombin in full explicit water, showing that after approximately 5 ns thrombin undergoes a significant conformational transition, from a more open conformation (which we propose can be related to the fast form) to a more compact and closed one (which we propose can be related to the slow form). This transition mainly involves the Trp148 and Trp60D loop, the S3 site, and the fibrinogen binding site, whereas the S1 site, the Na+-binding site, and the catalytic pocket remain essentially unchanged. In particular, our data indicate that the S3 site of the enzyme is less accessible to water in the putative slow form. This structural picture provides a reasonable molecular explanation for the fact that physiological substrates related to the procoagulant activity of thrombin (fibrinogen, thrombin receptor 1, and factor XIII) orient a bulky side chain into the S3 site of the enzyme. Taken together, our results can have important implications for the design of novel thrombin inhibitors, of practical utility in the treatment of coagulative disorders.     

Leishmania amazonensis exhibits phosphatidylserine-dependent procoagulant activity,a process that is counteracted by sandfly saliva

Leishmania parasites expose phosphatidylserine (PS) on their surface, a process that has been associated with regulation of host''s immune responses. In this study we demonstrate that PS exposure by metacyclic promastigotes of Leishmania amazonensis favours blood coagulation. L. amazonensis accelerates in vitro coagulation of human plasma. In addition, L. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. This process was reversed by annexin V which blocks PS binding sites. During blood meal, Lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. Since saliva and parasites are co-injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of L. amazonensis . Lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. It also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS vesicles or in L. amazonensis . Sandfly saliva inhibits factor X activation by the intrinsic tenase complex assembled on PC/PS vesicles and blocks factor Xa catalytic activity. Altogether our results show that metacyclic promastigotes of L. amazonensis are procoagulant due to PS exposure. Notably, this effect is efficiently counteracted by sandfly saliva.     

Proteolytic activity of alpha 2-macroglobulin-enzyme complexes toward human factor VIII/von Willebrand factor

The low level of enzymatic activity of certain alpha 2-macroglobulin-proteinase complexes could be important to the function of factor VIII/von Willebrand glycoprotein since it is especially sensitive to proteolytic cleavage. To test this possibility, complexes of alpha 2-macroglobulin with plasmin, trypsin, and thrombin were formed in at least a 2:1 molar ratio of alpha 2-macroglobulin:proteinase and tested for effects on the factor VIII procoagulant activity of the factor VIII/von Willebrand glycoprotein. Neither the alpha 2-macroglobulin-trypsin complex nor the alpha 2-macroglobulin-plasmin complex affected factor VIII procoagulant activity. The behavior of the alpha 2-macroglobulin-thrombin complex was different. When alpha 2-macroglobulin and thrombin were incubated in a mole ratio of 3:1 or less, factor VIII procoagulant activity was enhanced to about the same extent as with free thrombin. Even at a 24:1 mole ratio, the mixture could produce 45% of the increase in factor VIII activity obtained with free thrombin. The isolated alpha 2-macroglobulin-thrombin complex could also activate the factor VIII procoagulant function to about 45% of the level obtained with an identical amount of uncomplexed thrombin. Analysis of the alpha 2-macroglobulin-125I-labeled thrombin complexes by rechromatography or by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that this activation was not due to free thrombin. We conclude that the alpha 2-macroglobulin-thrombin complex retains sufficient proteolytic activity to activate the procoagulant function of factor VIII/von Willebrand glycoprotein despite the latter being a very large substrate, having an estimated molecular weight of 1-20 million.     

Anticoagulant activity of fucoidans from brown algae

The anticoagulant activity of polysaccharide fucoidans from 11 species of brown algae was studied. The anticoagulant activity was measured by the activated partial thromboplastin time (APTT), prothrombin time, and thrombin time. Inhibitory action of these fucoidans significantly varied from one species to another. Fucoidans from Laminaria saccharina and Fucus distichus exhibited high anticoagulant activity, while fucoidans from Cladosiphon okamuranus and Analipus japonicus were almost inactive. Other fucoidans exhibited intermediate inhibitory activity. The inhibitory effect of fucoidans on thrombin and factor Xa was investigated in the presence or in the absence of natural thrombin inhibitor, antithrombin III (AT III). In contrast to the best-studied anticoagulant, heparin, most of these fucoidans inhibited thrombin in the absence of AT III. In the presence of AT III the inhibitory effect of fucoidans considerably increased. In contrast to heparin, fucoidans weakly influenced factor Xa activity in the presence of AT III and their inhibitory effect was not observed in the absence of AT III. There was no correlation between the anticoagulant activities of this series of fucoidans and their anti-inflammatory action, studied earlier. It is suggested that these two types of fucoidan activities depend on different structural features of fucoidans. Results of this study demonstrate a possibility of preparation of fucoidans with high anti-inflammatory activity but low anticoagulant activity. Anticoagulant activity of the fucoidans did not exhibit direct dependence on the content of fucose, the other neutral sugars and sulfates; no dependence was also found between the anticoagulant activity and the structure of the backbone of their molecules.     

Thrombomodulin blocks the ability of thrombin to activate platelets

When thrombin is complexed to the endothelial cell surface receptor thrombomodulin, it loses its procoagulant activities in that it no longer clots fibrinogen or activates factor V. Studies were initiated to determine if complex formation also blocks thrombin's other major procoagulant function, the activation of platelets. When bound to thrombomodulin, thrombin no longer induces platelets to either aggregate or release [14C] serotonin. Binding studies using 125I-labeled thrombin or diisopropyl phosphorothrombin indicate that the complex does not bind to the platelet. When thrombomodulin is added after thrombin has bound to the platelets, the thrombin rapidly redistributes onto the thrombomodulin. These data suggest that in addition to its other anticoagulant effects, thrombomodulin may also act to inhibit and/or reverse platelet activation by thrombin.     

State of coagulation hemostasis and fibrinolysis processes in dynamics of rapidly progressing forms of botulinal intoxication

Phase disturbances of coagulation blood potential were revealed in experiments on white rats in dynamics of rapidly progressing form of botulinic intoxication, intoxication being caused by intraperitoneal injection of type C toxin. In preclinical period of intoxication activation of procoagulant and anticoagulant parts of hemostasis system, as well as fibrinolysis system, was noted. Similar shifts were revealed in the developmental period of generalized pareses of skeletal musculature. Only in the terminal stage of intoxication insufficiency of mechanism in formation of prothrombinase activity developed by simultaneous activation of anticoagulant mechanism and fibrinolysis system.     

Neutral glycosphingolipid-dependent inactivation of coagulation factor Va by activated protein C and protein S.

To test whether neutral glycosphingolipids can serve as anticoagulant cofactors, the effects of incorporation of neutral glycosphingolipids into phospholipid vesicles on anticoagulant and procoagulant reactions were studied. Glucosylceramide (GlcCer), lactosylceramide (LacCer), and globotriaosylceramide (Gb(3)Cer) in vesicles containing phosphatidylserine (PS) and phosphatidylcholine (PC) dose dependently enhanced factor Va inactivation by the anticoagulant factors, activated protein C (APC) and protein S. Addition of GlcCer to PC/PS vesicles enhanced protein S-dependent APC cleavage in factor Va at Arg-506 by 13-fold, whereas PC/PS vesicles alone minimally affected protein S enhancement of this reaction. Incorporation into PC/PS vesicles of GlcCer, LacCer, or Gb(3)Cer, but not galactosylceramide or globotetraosylceramide, dose dependently prolonged factor Xa-1-stage clotting times of normal plasma in the presence of added APC without affecting baseline clotting times in the absence of APC, showing that certain neutral glycosphingolipids enhance anticoagulant but not procoagulant reactions in plasma. Thus, certain neutral glycosphingolipids (e.g. GlcCer, LacCer, and Gb(3)Cer) can enhance anticoagulant activity of APC/protein S by mechanisms that are distinctly different from those of phospholipids alone. We speculate that under some circumstances certain neutral glycosphingolipids either in lipoprotein particles or in cell membranes may help form antithrombotic microdomains that might enhance down-regulation of thrombin by APC in vivo.     

Consumption of barley ameliorates the diabetic steatohepatitis and reduces the high transforming growth factor β expression in mice grown in α-minimum essential medium in vitro as embryos

Non-alcoholic fatty liver disease (NAFLD), which includes the subtype non-alcoholic steatohepatitis (NASH), is a major complication of type 2 diabetic mellitus (T2DM), even among non-obese patients. However, the exact cause of NAFLD/NASH in non-obese patients with T2DM is unclear. We studied a non-obese mouse model of T2DM created through the malnourishment of embryos by culture in vitro for 48 h in α-minimum essential medium (MEM) at the two-cell stage. We compared the development of steatohepatitis in these MEM mice with control mice that were similarly cultured in standard potassium simplex-optimized medium (KSOM). We also studied the effects of 10 weeks of consumption of barley, which contains large amounts of the soluble fiber β-glucan, on the steatohepatitis of the adult MEM mice. The size of lipid droplets, the area of fibrosis, and the mRNA expression of the transforming growth factor beta (Tgfb) gene in the liver were higher in adult MEM mice fed a rice-based diet than in KSOM mice fed the same diet. However, barley consumption reduced the area of fibrosis and TGFB expression in MEM mice. In conclusion, adult mice that are cultured in MEM at the two-cell embryo stage develop steatohepatitis and T2DM, accompanied by higher hepatic TGFB expression, than KSOM controls. Furthermore, the consumption of barley during adulthood ameliorates the steatohepatitis and reduces the TGFB expression.     

Expression of allosteric linkage between the sodium ion binding site and exosite I of thrombin during prothrombin activation

The specificity of thrombin for procoagulant and anticoagulant substrates is regulated allosterically by Na+. Ordered cleavage of prothrombin (ProT) at Arg320 by the prothrombinase complex generates proteolytically active, meizothrombin (MzT), followed by cleavage at Arg271 to produce thrombin and fragment 1.2. The alternative pathway of initial cleavage at Arg271 produces the inactive zymogen form, the prethrombin 2 (Pre 2).fragment 1.2 complex, which is cleaved subsequently at Arg320. Cleavage at Arg320 of ProT or prethrombin 1 (Pre 1) activates the catalytic site and the precursor form of exosite I (proexosite I). To determine the pathway of expression of Na+-(pro)exosite I linkage during ProT activation, the effects of Na+ on the affinity of fluorescein-labeled hirudin-(54-65) ([5F]Hir-(54-65)(SO-3)) for the zymogens, ProT, Pre 1, and Pre 2, and for the proteinases, MzT and MzT-desfragment 1 (MzT(-F1)) were quantitated. The zymogens showed no significant linkage between proexosite I and Na+, whereas cleavage at Arg320 caused the affinities of MzT and MzT(-F1) for [5F]Hir-(54-65)(SO-3) to be enhanced by Na+ 8- to 10-fold and 5- to 6-fold, respectively. MzT and MzT(-F1) showed kinetically different mechanisms of Na+ enhancement of chromogenic substrate hydrolysis. The results demonstrate for the first time that MzT is regulated allosterically by Na+. The results suggest that the distinctive procoagulant substrate specificity of MzT, in activating factor V and factor VIII on membranes, and the anticoagulant, membrane-modulated activation of protein C by MzT bound to thrombomodulin are regulated by Na+-induced allosteric transition. Further, the Na+ enhancement in MzT activity and exosite I affinity may function in directing the sequential ProT activation pathway by accelerating thrombin formation from the MzT fast form.     

组织因子阳性的细胞和微粒在胃癌高凝状态中的作用

目的:探讨胃癌患者血浆中组织因子阳性的血小板、白细胞和微粒的数量及其促凝活性。方法:将45例胃癌患者根据TNM分期分为Ⅰ、Ⅱ、Ⅲ、Ⅳ期,同时选取30例健康人作为对照组。采用流式细胞术检测组织因子阳性的细胞和微粒数。凝血酶生成实验检测细胞和微粒的凝血活性。结果:胃癌Ⅲ/Ⅳ期患者血浆中组织因子阳性的血小板、中性粒细胞、单核细胞和微粒的数量明显高于胃癌Ⅰ/Ⅱ期和健康对照组。胃癌Ⅲ/Ⅳ期患者血小板、白细胞和微粒的促凝活性与其他组相比显著升高,与增加凝血酶的生成速度和生成总量有关。用抗组织因子抗体抑制TF后,细胞和微粒的凝血活性明显下降。然而,使用抗膜连蛋白V抑制PS后,细胞和微粒的凝血活性虽然有下降趋势,但是并不明显。此外,根治性手术治疗可以降低组织因子阳性的血小板、中性粒细胞、单核细胞和微粒的数量。结论:组织阳性的血小板、中性粒细胞、单核细胞和微粒是胃癌Ⅲ/Ⅳ患者高凝状态的原因之一,通过抑制TF和凝血酶的生成可能降低胃癌患者的血栓发生率。     

Thrombomodulin enhances the invasive activity of mouse mammary tumor cells

Thrombomodulin (TM) is a thrombin receptor on the surface of endothelial cells that converts thrombin from a procoagulant to an anticoagulant. Thrombin promotes invasion by various tumor cells, and positive or negative correlations are found between the expression of TM and tumorigenesis in some patients. In this study, we used an invasion assay to investigate the effect of TM on the invasive activity of a mouse mammary tumor cell line, MMT cells, and the effects of TM were compared with those of thrombin as a positive control. In the presence of 1% fetal calf serum (FCS), TM significantly stimulated MMT cell invasion in a dose-dependent manner, resulting in an approximately 3-fold increase at 1-10 pg/ml over the untreated control. Thrombin also caused a similar degree of stimulation at 50 ng/ml. Since thrombin activity was detected in the components of the assay system, an invasion assay was also performed in a thrombin-activity-depleted assay system constructed to eliminate the effect of thrombin activity; TM (10 pg/ml) plus thrombin (1 pg/ml) stimulated invasion by approximately 3.5-fold in this assay system. Hirudin, a specific thrombin inhibitor, inhibited stimulation by TM as well as by thrombin in both the presence and absence of 1% FCS. Investigations of the effects of TM on proliferation, adhesion and chemotaxis to clarify the mechanism of stimulation by TM revealed that TM does not affect proliferation or adhesion in the presence of 1% FCS, but stimulates chemotaxis by approximately 2.3-fold. Similar results were obtained in experiments using thrombin. TM (10 pg/ml) plus thrombin (1 pg/ml), on the other hand, stimulated chemotaxis by approximately 2.3-fold in the thrombin-activity-depleted assay system. Binding studies using [125I]-thrombin revealed that the cells have specific saturable binding sites for thrombin. These results show that TM stimulates the invasive activity of MMT cells, probably by acting as a cofactor for the thrombin-stimulated invasion of the cells via its receptor and lowering the effective concentration of thrombin. The findings also indicate that the stimulation of invasive activity in the presence of 1% FCS and in the thrombin-activity-depleted assay system may mainly be mediated by the stimulation of chemotaxis.     

Rational design of a potent anticoagulant thrombin

Thrombin acts as a procoagulant when it cleaves fibrinogen and promotes the formation of a fibrin clot and functions as an anticoagulant when it activates protein C with the assistance of the cofactor thrombomodulin. The dual function of thrombin in the blood poses the challenge to turn the enzyme into a potent anticoagulant by selectively abrogating fibrinogen cleavage. Using functional and structural data, we have rationally designed a thrombin mutant, W215A/E217A, that cleaves fibrinogen with a value of k(cat)/K(m) about 20,000-fold slower than wild-type but activates protein C in the presence of thrombomodulin with a specificity comparable with wild-type. This mutant demonstrates for the first time that the relative specificity of thrombin toward fibrinogen and protein C can be completely reversed.     

Dissociation of activated protein C functions by elimination of protein S cofactor enhancement

Activated protein C (APC) plays a critical anticoagulant role in vivo by inactivating procoagulant factor Va and factor VIIIa and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor can initiate protease-activated receptor-1 (PAR-1)-mediated cytoprotective signaling. Protein S constitutes a critical cofactor for the anticoagulant function of APC but is not known to be involved in regulating APC-mediated protective PAR-1 signaling. In this study we utilized a site-directed mutagenesis strategy to characterize a putative protein S binding region within the APC Gla domain. Three single amino acid substitutions within the APC Gla domain (D35T, D36A, and A39V) were found to mildly impair protein S-dependent anticoagulant activity (<2-fold) but retained entirely normal cytoprotective activity. However, a single amino acid substitution (L38D) ablated the ability of protein S to function as a cofactor for this APC variant. Consequently, in assays of protein S-dependent factor Va proteolysis using purified proteins or in the plasma milieu, APC-L38D variant exhibited minimal residual anticoagulant activity compared with wild type APC. Despite the location of Leu-38 in the Gla domain, APC-L38D interacted normally with endothelial cell protein C receptor and retained its ability to trigger PAR-1 mediated cytoprotective signaling in a manner indistinguishable from that of wild type APC. Consequently, elimination of protein S cofactor enhancement of APC anticoagulant function represents a novel and effective strategy by which to separate the anticoagulant and cytoprotective functions of APC for potential therapeutic gain.     

Insight into the molecular basis of coagulation proteinase specificity by mutagenesis of the serpin antithrombin

Specific cleavage of factor V at several P1Arg sites is critical for maintenance of hemostasis. While cleavage by procoagulant proteinases fXa and thrombin activates the cofactor, its cleavage by the anticoagulant proteinase activated protein C (APC) inactivates it. Antithrombin (AT), a specific serpin inhibitor of both thrombin and factor Xa, but not APC, was used as a model system to investigate molecular determinants of APC specificity in the inactivation reaction. Two mutants were prepared in which the P2 or the P3-P3' residues of the reactive site loop of the serpin were replaced with the corresponding residues of the APC cleavage site in factor V spanning residues 504-509 (Asp(504)-Arg-Arg-Gly-Ile-Gln(509)). Kinetic analysis showed that the reactivities of mutants were impaired by approximately 2-3 orders of magnitude with both factor Xa and thrombin, but improved by approximately 2 orders of magnitude with APC. The saturable dependence of the observed first-order rate constants on the concentrations of AT in complex with approximately 70-saccharide high-affinity heparin revealed that changes in the reactivity of the 504-509 mutant with proteinases are primarily due to an effect in the second reaction step in which a noncovalent serpin-proteinase encounter complex is converted to a stable, covalent complex. These results suggest that the P3-P3' residues of the APC cleavage site in factor Va, particularly P2Arg, confer specificity for the anticoagulant proteinase by improving the reactivity of the catalytic pocket with the transition state of the substrate in the second step of the reaction.