Infusion of phospholipid vesicles amplifies the local thrombotic response to TNF and anti-protein C into a consumptive response.

Inflammation often is considered a contributing factor to both thrombosis and disseminated intravascular coagulation. The molecular mechanisms that dictate which of these clinical manifestations will result from the inflammatory stimulus remain obscure. Bacterial infection and certain tumors are common initiators of the disseminated intravascular coagulant response.

Protein C inhibitor is a potent inhibitor of the thrombin-thrombomodulin complex.

Protein C inhibitor (PCI), a plasma serine protease inhibitor, inhibits several proteases including the anticoagulant enzyme, activated protein C (APC), and the coagulation enzymes, thrombin and factor Xa. Previous studies have shown that thrombin and APC are inhibited at similar rates by PCI and that heparin accelerates PCI inhibition of both enzymes more than 20-fold. We now demonstrate that the thrombin-binding proteoglycan, rabbit thrombomodulin, accelerates inhibition of thrombin by PCI approximately equal to 140-fold (k2 = 2.

Cellular regulation of the protein C pathway.

The protein C anticoagulant pathway regulates thrombin formation. The pathway is triggered when thrombin binds to the endothelial cell proteoglycan, thrombomodulin. Unlike thrombin, this complex is a potent activator of the protein C zymogen, but it cannot clot blood.

Role of free protein S and C4b binding protein in regulating the coagulant response to Escherichia coli.

Previous studies showed that infusion of C4b-binding protein with sublethal Escherichia coli (E. coli) in the primate produced a consumptive coagulopathy followed by microvascular thrombosis and renal failure. The first objective of this study was to characterize the pathophysiology and mechanism of this phenomena following infusion of both these agents with emphasis on defining the role of free protein S.

Tryptophans 231 and 234 in protein C report the Ca(2+)-dependent conformational change required for activation by the thrombin-thrombomodulin complex.

Human protein C circulates as both single- and two-chain zymogens. Activation by the physiological activation complex, thrombin-thrombomodulin, generates the anticoagulant enzyme, activated protein C. Ca2+ binding to the protease domain of protein C is accompanied by 5.

Contribution of residue 192 in factor Xa to enzyme specificity and function.

Mutation of residue 192 (chymotrypsin numbering) from Glu to Gln in thrombin and activated protein C has been shown to dramatically alter substrate and inhibitor specificity, in large part by allowing these enzymes to accept substrates with acidic residues in the P3 and/or P3' positions. In factor Xa, residue 192 is already a Gln. We now compare the properties of a Q192E mutant of Gla-domainless factor X (GDFX).

An update on clinical and basic aspects of the protein C anticoagulant pathway.

The protein C anticoagulant pathway provides a mechanism for regulating the coagulation process through the selective inactivation of factors Va and VIIIa. Recent studies have suggested that factor V may facilitate this process and that a mutation at one of the inactivation sites can contribute to resistance to activated protein C (APC) inactivation of factor Va. This appears to be a common cause of familial thrombophilia.

Thrombomodulin as a model of molecular mechanisms that modulate protease specificity and function at the vessel surface.

The protein C anticoagulant system generates an "on demand" physiologic anticoagulant response. The pathway is initiated when thrombin binds to the endothelial cell thrombin binding protein, thrombomodulin. The complex exhibits dramatically altered macromolecular specificity.

Inflammation. They're not just for clots anymore.

The identification of the blood-clotting inhibitor, protein S, as a ligand for a previously 'orphan' family of receptor tyrosine kinases demonstrates a new role for clotting and anti-clotting proteins in regulating cell proliferation.

Factor Xa-factor Va complex assembles in two dimensions with unexpectedly high affinity: an experimental and theoretical approach.

The influence of phospholipid vesicle concentration and size on the affinity and the kinetics of assembly of the prothrombin activation complex are examined. Activation of prethrombin 1 was used to monitor complex formation between factors Va and Xa. When activation rates were measured immediately after the addition of the reactants, the rate of activation increased, and subsequently decreased, as a function of increasing vesicle concentration.

Molecular cloning and expression of murine and bovine endothelial cell protein C/activated protein C receptor (EPCR). The structural and functional conservation in human, bovine, and murine EPCR.

Recently, we identified and cloned a human endothelial cell protein C/activated protein C receptor (EPCR). EPCR was predicted to be a type 1 transmembrane glycoprotein and a novel member of the CD1/major histocompatibility complex superfamily with 28% identity with CD1d. Even greater homology (62% identity) was detected with the murine protein, CCD41, which was previously characterized as a centrosome-associated, cell cycle-dependent protein.

On the role of phosphatidylethanolamine in the inhibition of activated protein C activity by antiphospholipid antibodies.

Phosphatidylethanolamine (PE) is an important membrane component for supporting activated protein C anticoagulant activity but has little influence on prothrombin activation. This difference constitutes a potential mechanism for selective inhibition of the protein C anticoagulant pathway by lupus anticoagulants and/or antiphospholipid antibodies. In this study, we demonstrate that the presence of PE augments lupus anticoagulant activity.

Identification, cloning, and regulation of a novel endothelial cell protein C/activated protein C receptor.

Human protein C and activated protein C are shown to bind to endothelium specifically, selectively and saturably (Kd = 30 nM, 7000 sites per cell) in a Ca(2+)-dependent fashion. Expression cloning revealed a 1.3-kilobase pair cDNA that coded for a novel type 1 transmembrane glycoprotein capable of binding protein C.

Possible involvement of cytokines in diffuse intravascular coagulation and thrombosis.

Inflammation and the cytokines clearly affect the coagulation system. Less clear are the specific influences of the coagulation system on inflammation. In this chapter only some of the coagulation systems affected by the cytokines are discussed, and the influences on the fibrinolytic system, which is also downregulated by selected cytokines are not mentioned; see Schneiderman and Loskutoff (1991) for a brief review.

Asp-70-->Lys mutant of factor X lacks high affinity Ca2+ binding site yet retains function.

To study the role of a putative high affinity Ca2+ binding site in the protease domain of factor X, we prepared a deletion mutant (E2FX) lacking the Gla and first epidermal growth factor-like domains. E2FX possesses a single high affinity Ca2+ binding site (Kd = 154 microM). Asp-70 or Glu-80 (chymotrypsin numbering system) was then converted to Lys.

Calcium inhibition of the activation of protein C by thrombin. Role of the P3 and P3' residues.

Protein C, a precursor to a natural plasma anticoagulant, and the platelet thrombin receptor, involved in cell activation, both require proteolytic cleavage to be activated. In humans, the sequences adjacent to the scissile bond of protein C, DPR/LID and the thrombin receptor DPR/SFL are similar. Previous studies with Asp-->Gly mutants indicated that both the P3 and the P3' Asp residues make either peptides or protein C a poor substrate for free thrombin, but thrombin interaction with thrombomodulin overcomes these inhibitory interactions.

Glu192-->Gln substitution in thrombin yields an enzyme that is effectively inhibited by bovine pancreatic trypsin inhibitor and tissue factor pathway inhibitor.

Modeling studies have ascribed the remarkable resistance of thrombin to inhibition by the Kunitz type inhibitors, bovine pancreatic trypsin inhibitor (BPTI), and tissue factor pathway inhibitor (TFPI), to steric inhibition by the 60-loop insertion, especially Trp60D (in the chymotrypsin numbering system). Indeed, deletion of Pro60B, Pro60C, and Trp60D from this loop (des-PPW) enhances BPTI inhibition (Ki = 16 nM) (Le Bonniec, B. F.

Glycosaminoglycan contributions to both protein C activation and thrombin inhibition involve a common arginine-rich site in thrombin that includes residues arginine 93, 97, and 101.

Proteoglycans play pivotal roles in the regulation of thrombin. Thrombomodulin (TM) binds thrombin through protein-protein contacts and a chondroitin sulfate moiety. The complex activates the anticoagulant zymogen, protein C.

Lack of suppressed renal thrombomodulin expression in a septic rat model with glomerular thrombotic microangiopathy.

Background: The thrombomodulin-dependent protein C anticoagulant pathway plays a major physiologic role in the down-regulation of the coagulation process. In cell culture, inflammatory cytokines or endotoxin can down-regulate endothelial thrombomodulin (TM) suggesting that suppressed TM expression may contribute to thrombotic complications noted in Gram-negative sepsis.

Experimental Design: In the present study, we have examined TM expression in the kidneys of septic rats utilizing indirect immunofluorescence and have quantified TM antigen and TM activity in extracts of the same kidneys by enzyme-linked immunosorbent assays and protein C activation assays, respectively.

Proline at the P2 position in protein C is important for calcium-mediated regulation of protein C activation and secretion.

Protein C is a vitamin K-dependent plasma serine protease zymogen, which upon activation, functions as an anticoagulant. Protein C activation is catalyzed by a complex of thrombin (T) with thrombomodulin (TM). This activation is Ca(2+)-dependent, but Ca2+ inhibits protein C activation by thrombin alone.

Occupancy of anion binding exosite 2 on thrombin determines Ca2+ dependence of protein C activation.

Thrombomodulin (TM) binds thrombin to form a complex that activates the plasma anticoagulant zymogen protein C. TM is an integral membrane glycoprotein that contains a chondroitin sulfate moiety. Interaction with thrombin involves both the protein component of TM, specifically the growth factor-like repeats 4-6 (TM 4-6), and chondroitin sulfate.

Mapping of the thrombin des-ETW conformation by using site-directed mutants of hirudin. Evidence for the induction of nonlocal modifications by mutagenesis.

Deletion of Glu146, Thr147, and Trp148 from thrombin dramatically alters its interactions with substrates, ligands, and inhibitors, and the changes appear to result from nonlocal modification of thrombin's structure [Le Bonniec, B. F., Guinto, E.

Mutation of Glu-80-->Lys results in a protein C mutant that no longer requires Ca2+ for rapid activation by the thrombin-thrombomodulin complex.

Binding Ca2+ to a high affinity site in protein C and Gla-domainless protein C (protein C lacking residues 1-44) results in a conformational change that is required for activation by the thrombin-thrombomodulin complex, the natural activator of protein C. Protein C modeling studies suggested the single high affinity Ca2+ binding-site might be present in a loop in the protease domain and involve Glu-70 and -80 (chymotrypsin numbering system). This loop, which is a known Ca(2+)-binding site in trypsin, is also conserved in other coagulation proteases, including factors VII, IX,and X.

Phosphatidylethanolamine incorporation into vesicles selectively enhances factor Va inactivation by activated protein C.

Membrane surfaces accelerate the proteolytic inactivation of factor Va by activated protein C. In most coagulation complexes, the most active membrane phospholipid is believed to be phosphatidylserine. In this study, we demonstrate that with phosphatidylserine-containing vesicles, incorporation of phosphatidylethanolamine increased the rate of factor Va inactivation approximately 10-fold at all concentrations of factor Va studied and at all vesicle concentrations at or below the optimum for prothrombin activation.