Calibrated automated thrombography displays the concentration of thrombin in clotting plasma with or without platelets (platelet-rich plasma/platelet-poor plasma, PRP/PPP) up to 48 samples by monitoring splitting a fluorogenic substrate and comparing it constant known activity parallel, non-clotting sample. Thus, non-linearity reaction rate is compensated for, adding an excess can be avoided. Standard conditions were established at which acceptable experimental variation accompanies...

The kinetic parameters of the conversion bovine prothrombin into thrombin by activated blood clotting factor X (X,) have been determined in absence and presence Ca", V (V,,), phospholipid (dioleoylphosphatidylcholine/ dioleoylphosphatidylserine, 1: 1; mol/mol).In accessory components, K , for is 131 F, which well above its concentration plasma about 1.5 PM.The Vmax formation 0.61 mol min" X,-' under these conditions.In 7.5 PM phospholipid, drops to 0.058 V,, slightly increases

Vascular anticoagulant alpha (VAC alpha, annexin V) is a member of the family calcium and phospholipid binding proteins, annexins. The properties VAC to bilayers were studied by ellipsometry. Adsorption was calcium-dependent completely reversible upon depletion. Half-maximal adsorptions consisting 100, 20, 5, 1% dioleoyl-phosphatidylserine (DOPS) supplemented with dioleoyl-phosphatidylcholine (DOPC) reached at Ca2+ concentrations 0.04, 0.22, 1.5, 8.6 mM. These surfaces all showed same...

The kinetic parameters of bovine factor X activation by I , have been determined in the absence and presence Ca*+, thrombin-activated VIII (vnr,), phospholipid (dioleoylphosphatidylcholine/dioleoylphosphatidylserine, 75/25; mol/ mol).Factor Ca2+, VIII,, is able to catalyze activation.The X,,, for 299 p~ which well above its concentration plasma, about 0.2 p ~.The V,, X, formation 0.0022 mol X.-min" .mol under these conditions.Addition Ca2+ has little effect on constants D L .In 10 m~ CaCL...

In the present paper we compare prothrombin‐converting activities of platelets non‐activated, or activated by collagen, thrombin collagen plus in absence and presence added factor V a . all experiments described, rate formation for combined action is greater than that stimulated alone. The enhanced cases, but higher activity observed with remains. When are X prothrombin, lag period approximately 10 min before reaches steady state. Addition an excess this experiment reduces time to 3 min....

Summary By the use of a fluorogenic thrombin substrate and continuous calibration each individual sample, it is now possible to obtain generation (TG) curve (or thrombogram) in plasma, with or without platelets, an easy routine procedure at high throughput acceptable experimental error (< 5%). Evidence growing that parameters thrombogram, notably area under (endogenous potential, ETP), are useful assessing bleeding-or thrombotic risk its modification by antithrombotic-or haemostatic...

The murine/human chimeric monoclonal antibody fragment (c7E3 Fab) blocks GPIIb/IIIa and alpha v beta 3 receptors, inhibits platelet aggregation, decreases the frequency of ischemic events after coronary artery angioplasty in patients at high risk suffering such events. Although inhibition aggregation is likely to be major mechanism c7E3 Fab's effects, since activated platelets facilitate thrombin generation, it possible that Fab also generation. To test this hypothesis, effects other...

We investigated by means of an automated ellipsometer the adsorption prothrombin from a buffer solution multilayers 14:0/14:0-and 18:1/18:1phosphatidylserine (PS) stacked on chromium slides.In this instrument thickness and refractive index adsorbed phospholipid proteins are monitored continuously.Two equations derived to relate mass phospholipids protein index.These based upon Lorentz-Lorenz relation among molar refractivities, indices, densities binary mixtures.Experimental validation these...

Epidemiological studies have shown that women who use third-generation oral contraceptives (OC) containing desogestrel, gestodene or norgestimate a higher risk of venous thrombosis than second-generation OC levonorgestrel. It is also known mutation in factor V (factor V(Leiden)), which results resistance to activated protein C (APC) and the most common cause hereditary thrombophilia, potentiates prothrombotic effect OC. Effects APC on thrombin generation plasma using were compared response...

A method is described by which the time-course of thrombin generation in plasma can be obtained from a continuous optical density recording p-nitroaniline (pNA) production 2:3 diluted plasma. chromogenic substrate, methylmalonyl-methylanalyl-arginyl-pNA (SQ 68), used that specifically split but at low rate. The appears and disappears does not more than 5% substrate added, so rate conversion good approximation proportional to amidolytic activity over entire period generation. course enzyme...

Summary In haemophilia patients, a relationship is usually observed between the clinical expression of disease and plasmatic factor VIII/factor IX (FVIII/FIX) activity. However, it known from experience, that some despite similar FVIII/FIX plasma levels, could exhibit different bleeding phenotype. After determining preanalytical test conditions, we evaluated thrombin generation capacity samples in various conditions potential usefulness (TGT) patients. series 46 patients (34 A 12 B...

Thrombin generation is a pivotal function of plasma in haemostasis and thrombosis. Its mechanism essentially the classical cascade, velocity which governed by availability factors Va VIIIa that confined to surface procoagulant membranes appear at site injury. There no routine test quantitatively renders thrombin forming capacity sample. Clotting times (PT-prothrombin time, APTT-activated partial tromboplastin time) do not reflect over all are insensitive hypercoagulative states. The...

A method is described in which thrombin activity clotting plasma can be monitored through the continuous measurement of fluorescent split-product substrate Z-Gly-Gly-Arg-AMC. The signal not impaired by turbidity; therefore proper disturbed occurrence a clot or presence platelets and direct platelet rich possible.

We investigated the relation between clotting factor concentration, parameters of thrombin generation curve (the thrombogram) and severity clinically observed bleeding in patients with congenital deficiency prothrombin (n = 21), V 22), VII X 10), XI 7) XII 6). The used were: area under (endogenous potential, ETP), peak concentration attained lag time before manifest formation. Peak height ETP varied linearly prothrombin. For other factors these hyperbolically approached to 100% limit...

A method is described that, on the basis of time course amidolytic activity after triggering thrombin generation in normal plasma, allows calculation velocity prothrombin conversion independent inactivating processes. It shown how reaction constants for alpha 2M-dependent and 2M-independent inactivation processes can be obtained a sample whole plasma. The verified by demonstrating that experimentally observed courses residual 2M-thrombin complex coincide with those calculated from activity,...

Factor V was purified from the plasma of an activated protein C (APC)-resistant patient who is homozygous for mutation Arg506→ Gln (factor VR506Q). VR506Q converted by thrombin into factor Va which further yielding a preparation that had same cofactor activity in prothrombin activation as normal Va. Inactivation low concentrations (<5 nM) 0.15 nM APC presence phospholipid vesicles proceeded via biphasic reaction consisted rapid phase (k = 4.3 × 107M−1s−1), intermediate with reduced fully...

Summary The influence of heparin on prothrombin conversion in plasma was estimated by measuring disappearance with the aid staphylocoagulase or calculation from thrombin generation curve, compensating for simultaneous inactivation protease inhibitors. In thromboplastin-activated effect prothrombinase (factor Xa-factor Va-phospholipid) is negligible compared to that probably because both extrinsic factor X activating complex and phospholipid bound Xa are insensitive AT III-heparin action....

In 1985 we isolated a new vascular anticoagulant protein VAC alpha, now called annexin V, with high binding affinity (Kd less than 10(-10) M) for phospholipids. Its effect was attributed to displacement of coagulation factors from the phospholipid membrane. The present study demonstrates that inhibition prothrombinase activity by V strongly depends on curvature membrane surface and calcium concentration. Half-maximal small vesicles, composed 20% phosphatidylserine 80% phosphatidylcholine,...

An anticoagulant fraction was isolated from the homogenate of human umbilical cord arteries, using Sephadex gel filtration and DEAE-Sephacel chromatography. Analysis with dodecyl sulfate/polyacrylamide electrophoresis inactivation studies proteolytic enzymes indicate that activity is associated a polypeptide an apparent Mr 32000. The inhibits thromboplastin as well factor Xa induced clotting but does not affect thrombin initiated fibrin formation. activation prothrombin by complete...

Proinflammatory effects induced by the serine protease factor Xa were investigated in HUVEC. Exposure of cells to (5-80 nM) concentration dependently stimulated production IL-6, IL-8, and monocyte chemotactic protein-1 (MCP-1) expression E-selectin, ICAM-1, VCAM-1, which was accompanied polymorphonuclear leukocyte adhesion. The blocked antithrombin III, but not thrombin-specific inhibitor hirudin, suggesting that elicits these responses directly via thrombin. IL-1alpha TNF-alpha implicated,...