Initiating and potentiating role of platelets in tissue factor‐induced thrombin generation in the presence of plasma: subject‐dependent variation in thrombogram characteristics

Abstract

Summary. The hemostatic activity of plasma is determined by platelet activation and coagulation, which processes are mutually stimulatory. We studied this interaction by measuring the cleavage of fluorescent thrombin substrate in platelet-rich plasma (PRP), using the calibrated thrombogram method. In freshly isolated human plasma, thrombin formation triggered by tissue factor was fully dependent on the presence of platelets. It was abolished by annexin A5, indicating dependence on phosphatidylserine (PS) exposure at activated platelets. Comparison of plasmas from various subjects showed considerable interindividual variation in total amount of thrombin generation, regardless of whether platelets or PS-containing phospholipids were present. Integrin a IIb b 3 antagonists and ADP receptor blockage, but not aspirin, decreased the rate of thrombin generation (thrombin peak level) and extended the time of onset. Platelet inhibition with cAMP-elevating agents decreased the thrombin-forming rate, but surprisingly shortened the onset time. Stimulation of platelets with agonists of Gi/q-coupled receptors and, to a larger extent, with collagen or Ca 2+ -ionophore increased the rate of thrombin generation and shortened its onset. In PRP from donors with low and high generation, platelet inhibitors and activators were similarly effective. Taken together, these results indicate that, in tissue factor-triggered PRP, PS exposure on activated platelets regulates both onset and rate of thrombin generation. However, coagulant activity rather than platelet activation determines the total amount of thrombin formed, i.e. the endogenous thrombin potential. Thus, kinetics of thrombin generation in PRP are controlled by platelet inhibitors and agonists, but the process is restricted in amount by the subject-dependent variation in coagulation.

Thrombin generation in mesalazine refractory ulcerative colitis and the influence of low molecular weight heparin

“…The study demonstrates that the thrombin potential, both intrinsic and extrinsic, is above the norm in patients with active UC, confirming more indirect data on hypercoagulation in these patients [16]. The administration of LMWH diminished the ITP and ETP in patients with UC, comparative to the effect of LMWH on patients with active thrombo-embolic diseases [18,30,32].…”

“…The ETP and ITP level is expressed as a percentage of the ETP and ITP of normal pool plasma, measured simultaneously. The mean value of ETP in PPP in healthy controls was 737 nmol/l min (range 720-790), the mean ITP level in PPP in healthy controls was 798 nmol/l min (range 780-850) [29,30]. The normal values of ETP, ITP, as well as TAT and F1.2, were obtained from ten healthy controls (five male, five female), and were compared with the values of the patients.…”

Lack of bleeding in patients with severe factor VII deficiency

“…Plateletrich plasma (PRP) and platelet-poor plasma (PPP) were prepared, and the preparations were studied in the Thromboscope TM System (Thromboscope, Maastrich, Netherlands). In this system, described in detail elsewhere [3,4], clotting is initiated using a small volume (20 mL) of recombinant tissue factor as well as CaCl 2 (0.1 M, 20 mL) and the subsequent thrombin generated detected using a fluorogenic substrate (Z-GGR-AMC, 2.5 mM) in a Fluoroskan Ascent well plate reader (Thermolab Systems, Helsinki, Finland). A calibrator is used to determine the amount of thrombin generated.…”

Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4

“…The observation that ETP was modestly influenced by increasing concentrations of histones might be explained by its lower sensitivity to platelet activation with respect to the other parameters. 31,32 The effect of histones was entirely platelet dependent, as shown by the following observations: (1) histones failed to induce detectable thrombin formation in CTI-inhibited PPP even in the presence of an exogenous procoagulant surface, indicating that they are not able to trigger thrombin generation by themselves; and (2) histone-induced thrombin generation in CTI-inhibited PRP was strongly reduced by several agents that interfere with platelet function at different levels, implying that the histone effect requires cellular signaling. These findings raise the question of how histone-activated platelets can trigger blood coagulation in CTI-inhibited PRP.…”