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The influence of platelets on tissue factor (TF)-initiated thrombin generation in a reconstituted model of blood coagulation and in whole blood was evaluated. No thrombin generation was observed over 15 min in the reconstituted model when either TF or platelets and phospholipids were omitted. At 25 pM TF, the rates of thrombin generation were platelet and PCPS concentration-dependent and achieved maximum (1.0 nM/s) in the physiological range of platelet concentration. Similar rates were achieved in the absence of platelets when 1-2 microM phospholipid was used. However, the maximum rates of thrombin generation (5.2-6.0 nM/s) and the shortest initiation phase (1 min) were attained between 25 and 100 microM phospholipid. In the reconstituted model, an increase in platelet concentration from 0.125 x 10(8)/ml to 0.5 x 10(8)/ml decreased the duration of the initiation phase (in the absence of phospholipids) from 4.3 min to 2 min. Further increases in platelet concentration did not affect this phase. Sequential whole blood studies were conducted in blood of a chemotherapy patient who developed reduced platelet counts. The TF (12.5 pM) initiated clotting of patient's blood was accelerated from approximately 10 min to 5 min when the platelet concentration increased from 0.05 x 10(8)/ml to 0.11 x 10(8)/ml. Clotting times were essentially unchanged for platelet concentrations exceeding 0.5 x 10(8)/ml (range 0.5-3.1 x 10(8)/ml). Similarly, clotting of whole blood obtained from healthy volunteers was not affected by the platelet count, which varied from 1.5 x 10(8)/ml to 3.1 x 10(8)/ml (4.0+/-0.5 min). The data obtained in both models are consistent with in vivo observations that clinical bleeding is most likely to occur at platelet counts

Apoptosis and chemokine induction after renal ischemia-reperfusion. Daemen MA, de Vries B, van't Veer C, Wolfs TG, Buurman WA. Department of General Surgery of the University of Maastricht, The Netherlands. BACKGROUND: One of the earliest prerequisites for the development of inflammation after ischemia-reperfusion (I/R) is local chemokine expression. We recently demonstrated that apoptosis, characterized by intracellular caspase-activation, contributes to the development of inflammation after I/R. METHODS: The contribution of apoptosis was investigated using the pan-caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2F in a murine model of renal I/R. Renal expression of the chemokines macrophage inflammatory protein-2 (MIP-2) and KC was studied using RT-PCR and immunohistology. Measuring myeloperoxidase activity and serum ureum and creatinine levels assessed neutrophil influx and kidney dysfunction. RESULTS: We demonstrate renal up-regulation of KC and MIP-2 after 1 to 16 hr of reperfusion. Treatment with the caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2F effectively prevented I/R-induced renal apoptosis, KC, and MIP-2 up-regulation after 2 hr of reperfusion as well as neutrophil influx and functional impairment after 24 hr of reperfusion. CONCLUSIONS: These data for the first time show that chemokine induction following I/R is dependent on caspase activation

Tissue injury as a consequence of ischemia followed by reperfusion is characterized by early as well as late signs of inflammation. The latter, among others, involves IFN-gamma-dependent up-regulation of MHC class I and II Ag expression. Employing a murine model of renal ischemia, we show that renal IL-18 mRNA up-regulation coincides with caspase-1 activation at day 1 following ischemia. IFN-gamma and IL-12 mRNA are subsequently up-regulated at day 6 following ischemia. Combined, but not separate, in vivo neutralization of the IFN-gamma inducing cytokines IL-12 and IL-18 reduces IFN-gamma-dependent MHC class I and II up-regulation to a similar extent as IFN-gamma neutralization, suggesting the involvement of functional IL-12, IL-18, and IFN-gamma protein. These results reveal a novel relationship between tissue injury of nonmicrobial origin and the induction of IL-12 as well as IL-18. The collaboration observed between endogenous IL-12 and IL-18 in the induction of IFN-gamma after renal ischemia/reperfusion, resembles the immune response to bacterial infections

Proinflammatory effects induced by the serine protease factor Xa were investigated in HUVEC. Exposure of cells to factor Xa (5-80 nM) concentration dependently stimulated the production of IL-6, IL-8, and monocyte chemotactic protein-1 (MCP-1) and the expression of E-selectin, ICAM-1, and VCAM-1, which was accompanied by polymorphonuclear leukocyte adhesion. The effects of factor Xa were blocked by antithrombin III, but not by the thrombin-specific inhibitor hirudin, suggesting that factor Xa elicits these responses directly and not via thrombin. IL-1alpha and TNF-alpha were not implicated, since neither the IL-1 receptor antagonist nor a TNF-neutralizing Ab could suppress the factor Xa responses. Active site-inhibited factor Xa and factor Xa depleted from gamma-carboxyglutamic acid residues were completely inactive. The effector cell protease receptor-1 (EPR-1) seems not to be involved since anti-EPR-1 Abs failed to inhibit cytokine production. Moreover, neither the factor X peptide Leu83-Leu88, representing the inter-epidermal growth factor sequence in factor Xa that mediates ligand binding to EPR-1, nor the peptide AG1, corresponding to the EPR-1 sequence Ser123-Pro137 implicated in factor Xa binding, inhibited the factor Xa-induced cytokine production. In conclusion, these findings indicate that factor Xa evokes a proinflammatory response in endothelial cells, which requires both its catalytic and gamma-carboxyglutamic acid-containing domain. The receptor system involved in these responses induced by factor Xa remains to be established.

Proinflammatory effects induced by the serine protease factor Xa were investigated in HUVEC. Exposure of cells to factor Xa (5-80 nM) concentration dependently stimulated the production of IL-6, IL-8, and monocyte chemotactic protein-1 (MCP-1) and the expression of E-selectin, ICAM-1, and VCAM-1, which was accompanied by polymorphonuclear leukocyte adhesion. The effects of factor Xa were blocked by antithrombin III, but not by the thrombin-specific inhibitor hirudin, suggesting that factor Xa elicits these responses directly and not via thrombin. IL-1alpha and TNF-alpha were not implicated, since neither the IL-1 receptor antagonist nor a TNF-neutralizing Ab could suppress the factor Xa responses. Active site-inhibited factor Xa and factor Xa depleted from gamma-carboxyglutamic acid residues were completely inactive. The effector cell protease receptor-1 (EPR-1) seems not to be involved since anti-EPR-1 Abs failed to inhibit cytokine production. Moreover, neither the factor X peptide Leu83-Leu88, representing the inter-epidermal growth factor sequence in factor Xa that mediates ligand binding to EPR-1, nor the peptide AG1, corresponding to the EPR-1 sequence Ser123-Pro137 implicated in factor Xa binding, inhibited the factor Xa-induced cytokine production. In conclusion, these findings indicate that factor Xa evokes a proinflammatory response in endothelial cells, which requires both its catalytic and gamma-carboxyglutamic acid-containing domain. The receptor system involved in these responses induced by factor Xa remains to be established

Three model systems have been used to study the dynamics of the blood clotting process initiated by tissue factor (TF): synthetic plasma mixtures prepared with purified coagulation proteins and inhibitors; mathematical models based on the reaction constants, stoichiometries and thermodynamics of individual catalyst and inhibitor reactions; and contact suppressed whole blood induced to clot in vitro by the addition of exogenous TF. In the three models, the generation of thrombin can be described in terms of an initiation phase in which pmol/l concentrations of the coagulation serine proteases are generated and the cofactor proteins factor V (FV) and FVIII are activated. Subsequently, explosive thrombin generation occurs during a propagation phase. The complementary inhibitory pathways extinguish the generation of thrombin. Tissue factor pathway inhibitor (TFPI), present in low concentrations, primarily influences the duration of the initiation phase and has little influence on the propagation phase. Antithrombin III (ATIII), present in higher concentrations, has little influence during the initiation phase, but decreases the rate of thrombin generation during the propagation phase. The protein C pathway cannot act in the absence of thrombin and therefore only influences the duration of the propagation phase by inactivating activated FV. Thus combinations of TFPI plus ATIII and TFPI plus protein C pathway components contribute to the synergistic inhibitory processes. As a consequence of the roles of pro, and anti-coagulants, the generation of thrombin by the TF pathway becomes a threshold limited process

Blood coagulation is initiated following damage of the vessel wall. The large number of proteins that participate in the reactions that regulate blood coagulation must behave in a concerted manner to generate alpha-thrombin. This most abundant enzyme product of the coagulation process acts on the soluble blood protein fibrinogen to create the insoluble fibrin clot, trigger platelet activation, and initiate a variety of vascular processes ranging from coagulation inhibition and activation of fibrinolysis to cell growth. The majority of enzymes that participate in the blood-clotting process circulate in blood as inactive zymogens and procofactors. Following mechanical injury to the vessel wall, a cell-associated protein, tissue factor, is exposed and initiates a series of enzymatic reactions localized on a membrane surface generally provided by cells and accumulating platelets. The binding of the enzymes and zymogen substrates to the exposed membrane surface and their activation are in part governed by the gamma-carboxyglutamic acid residues (gla) of each protein. Recently, it has been demonstrated that the gla residues are necessary for Ca2+ binding and subsequent exposure of the hydrophobic membrane binding regions to the cell bilayer. Thus, the gla residues play a preeminent role during the blood-clotting process. Absence of gamma-carboxylation of one or more of the critical glutamic acid residues results in an impaired coagulation/anticoagulation process which may lead to a bleeding diathesis or thrombosis

Protein S is a vitamin K-dependent nonenzymatic coagulation factor involved in the regulation of activated protein C (aPC). In this study, we report an aPC-independent anticoagulant function of protein S in plasma under flow conditions. Plasma, anticoagulated with low-molecular-weight heparin allowing tissue factor-dependent prothrombin activation, was perfused at a wall shear rate of 100 s-1 over tissue factor containing matrices of stimulated endothelial cells placed in a perfusion chamber. Fractions were collected in time at the outlet and prothrombin activation was determined by measuring the activation fragment F1+2 of prothrombin. In normal plasma, a time-dependent prothrombin activation was detected by the generation of fragment1+2. Prothrombin activation had ceased after 12 minutes perfusion, independent of the amount of tissue factor present in the matrix. Depletion of protein S from plasma or inhibition of protein S in plasma by monoclonal antibodies induced a 5- to 25-fold increase of prothrombin activation on the procoagulant endothelial cell matrix. A prolonged prothrombin activation was detected in protein S-depleted plasma up to 20 minutes after onset of the thrombin generation. The increased prothrombin activation in protein S-depleted plasma could not be explained by the absence of the cofactor function of protein S for aPC because depletion of protein C from plasma did not result in increased prothrombin activation. These data provide further evidence for a strong anticoagulant function of protein S in plasma independent from activated protein C.