Tissue factor pathway.

Blood coagulation is initiated in response to vessel damage in order to preserve the integrity of the mammalian vascular system. The coagulation cascade can also be initiated by mediators of the inflammatory response, and fibrin deposition has been noted in a variety of pathological states. The cascade of coagulation zymogen activations which leads to clot formation is initiated by exposure of flowing blood to Tissue Factor (TF), the cellular receptor and cofactor for Factor VII (FVII). FVII binds to the receptor in a I:I stoichiometric complex and is rapidly activated. FVIIa undergoes an active site transition upon binding TF in the presence of calcium which enhances the fundamental properties of the enzyme. This results in rapid autocatalytic activation of FVII to FVIIa, thereby amplifying the response by generating more TF-FVIIa complexes. The TF-FVIIa activates both FIX and FX. Further FXa generation by the FIXa-FVIIIa-Ca2+-phospholipid complex is required to sustain the coagulation mechanism, since the TF-FVIIa complex is rapidly inactivated by Tissue Factor pathway inhibitor (TFPI). TFPI circulates in plasma, is associated with vascular cell surface and is released from platelets following stimulation by thrombin. TFPI requires the formation of an active TF-FVIIa complex and FXa generation before inhibition can occur. TFPI prevents further participation of TF in the coagulation process by forming a stable quaternary complex, TF-FVIIa-FXa-TFPI.     

Endothelium-derived relaxing factor modulates platelet aggregation in an in vivo model of recurrent platelet activation.

It has been shown that endothelium-derived relaxing factor (EDRF) may inhibit platelet aggregation in vitro through activation of platelet-soluble guanylate cyclase. To assess whether EDRF may also affect platelet function in vivo, intravascular platelet aggregation was initiated by placing an external constrictor around endothelially injured rabbit carotid arteries. Carotid blood flow velocity was measured continuously by a Doppler flow probe placed proximal to the constrictor. After placement of the constrictor, cyclic flow reductions (CFRs), due to recurrent platelet aggregation, developed at the site of the stenosis. After CFRs were observed for 30 minutes, a solution of authentic nitric oxide (NO, n = 10) was infused into the carotid artery via a small catheter placed proximally to the stenosis. Before infusion of NO, CFR frequency averaged 18.3 +/- 2.9 cycles per hour, and CFR severity (lowest carotid blood flow as percentage of baseline values) was 6 +/- 1%. NO completely inhibited CFRs in all animals, as shown by the normal and constant pattern of carotid blood flow (CFR frequency, 0 cycles per hour, p < 0.001; carotid blood flow, 92 +/- 5%, p = NS versus baseline). These effects were transient; CFRs were restored spontaneously within 10 minutes after cessation of NO infusion. After CFRs returned, S-nitroso-cysteine (S-NO-cys), a proposed form of EDRF, was infused into the carotid artery. S-NO-cys also abolished CFRs in all animals but at a significantly lower dose than NO (0.3 +/- 0.1 versus 12 +/- 4 nmol/min). The role of endogenously released EDRF in modulating in vivo platelet function was then tested in additional experiments. In 10 animals, endogenous release of EDRF was stimulated by infusing acetylcholine into the aortic root during CFRs. Infusion of acetylcholine was also associated with a complete inhibition of CFRs, similar to that observed during exogenous infusion of NO or S-NO-cys. These antithrombotic effects of acetylcholine were completely lost when EDRF synthesis was prevented by administration of the L-arginine analogue NG-monomethyl L-arginine (L-NMMA). Furthermore, in six additional rabbits the basal release of EDRF was blocked by L-NMMA after CFRs had been previously abolished with aspirin or the combination of aspirin and ketanserin, a serotonin S2 receptor antagonist. L-NMMA caused restoration of CFRs in all animals, indicating that even the basal release of EDRF is important in modulating platelet reactivity in vivo. Taken together, the data of the present study demonstrate that endogenous EDRF might importantly contribute to the modulation of platelet function in vivo.     

Plasma levels of beta-thromboglobulin and platelet factor 4 as indices of platelet activation in vivo

Measurement of plasma levels of two secreted platelet proteins (beta- thromboglobulin and platelet factor 4) has been suggested as a means for detecting increased platelet activation in vivo. A crucial question in the measurement is the distinction between in vivo and in vitro secretion of the proteins. One approach to this distinction is the measurement of both proteins in each sample. These proteins are present in platelets in similar amounts and are released in similar quantities, but the plasma levels of beta-thromboglobulin exceed the plasma levels of platelet factor 4. This difference in plasma level is presumably due to more rapid removal of platelet factor 4 from the plasma level, and there is suggestive evidence that the rapid removal of released platelet factor 4 is due to its binding to endothelial cells. It appears that when there is increased release of beta-thromboglobulin and platelet factor 4 in vivo, there is an increase in the ratio of plasma beta-thromboglobulin to plasma platelet factor 4 compared to that found in normal individuals, whereas when in vitro release is responsible for elevated levels, the ratio decreases. Thus measurements of both proteins in each blood sample will allow distinction between in vivo release and artefactual in vitro release.     

Tissue factor pathway inhibitor endocytosis

Tissue factor pathway inhibitor (TFPI), a 42 kD protein, provides the physiological inhibition of tissue factor initiated coagulation by inhibition of both factor Xa and factor VIIa/tissue factor. In plasma, most TFPI is lipoprotein bound with an additional "releasable" pool bound to the endothelial cell surface. TFPI clearance is via receptor mediated endocytosis into liver. Heparin sulfate proteoglycans and LRP (low density lipoprotein receptor-related protein), an extremely large (～600 kD) cell surface protein, primarily mediate this clearance, although additional TFPI binding sites and endocytosis pathways exist. (Trends Cardiovasc Med 1997; 7:234-239). ? 1997, Elsevier Science Inc.     

Collagen-platelet interaction: Gly-Pro-Hyp is uniquely specific for platelet Gp VI and mediates platelet activation by collagen.

OBJECTIVE: Peptides consisting of a repeat Gly-Pro-Hyp sequence are potent platelet agonists. The aim of this study was: (1) to examine the specificity of this sequence for platelet activation; (2) to confirm its recognition by platelet glycoprotein VI; and (3) to assess with suitable peptides the relative importance of glycoprotein VI and integrin alpha 2 beta 1 in platelet activation by collagen. METHODS: Peptides were synthesized by standard Fmoc chemistry and tested for their ability to support adhesion of human platelets and HT 1080 cells, induce platelet aggregation, bind integrin alpha 2 subunit A-domain and to cause tyrosine phosphorylation of platelet proteins. RESULTS: (1) Peptides consisting of a repeat Gly-Pro-Pro, Gly-Pro-Ala or Gly-Pro-Arg sequence exhibited little if any platelet-reactivity. (2) The platelet-reactive peptide consisting of a repeating Gly-Pro-Hyp sequence failed to induce tyrosine phosphorylation in glycoprotein VI-deficient platelets. Platelet adhesion to this peptide was inhibited by intact anti-glycoprotein VI antibody and its Fab fragment. The latter inhibited aggregation by the peptide and fibres of both collagens I and III. (3) A peptide containing a 15-mer alpha 2 beta 1-binding sequence in a repeat Gly-Pro-Pro structure supported alpha 2 beta 1-mediated platelet and HT 1080 cell adhesion and bound alpha 2 A-domain, but failed to activate platelets or to induce tyrosine phosphorylation. Conversely, a peptide containing this sequence but with an essential Glu replaced by Ala and inserted in a repeat Gly-Pro-Hyp structure did not recognize alpha 2 beta 1, but was highly platelet activatory. CONCLUSIONS: Platelet activation by collagen involves the highly-specific recognition of the Gly-Pro-Hyp sequence by platelet glycoprotein VI. Recognition of alpha 2 beta 1 is insufficient to cause activation. Interaction between collagen and glycoprotein VI is unique since Gly-Pro-Hyp is common in collagens but occurs rarely in other proteins, and glycoprotein VI may be expressed solely by platelets. This sequence could provide a basis for a highly-specific anti-thrombotic reagent to control thrombosis associated with plaque rupture.     

In vivo platelet activation in mild arterial hypertension

In order to investigate whether an altered "in vivo" platelet serotonin release contributes to the low platelet serotonin content observed in essential hypertensive patients, we have measured plasma concentrations of beta-thromboglobulin and platelet factor 4, urinary beta-thromboglobulin concentrations and platelet serotonin and platelet factor 4 contents in 11 untreated essential hypertensive patients (WHO Stage I) and in 12 age-matched normotensive controls. Beta-thromboglobulin and platelet factor 4 are specific platelet proteins localized in the alpha-granules which are released during in vivo platelet activation. Plasma and urinary concentrations of these alpha granule proteins determined by radioimmunoassay were found to be similar in hypertensive and normotensive subjects. The low platelet serotonin content determined by high pressure liquid chromatography from platelet rich plasma in hypertensive patients (0.282 +/- 0.008 vs 0.348 +/- 0.019 nmol/10(8) platelets, p less than 0.01) was not associated with a decrease in platelet 4 content (1.36 +/- 0.07 vs 1.36 +/- 0.10 microgram/10(8) platelets). This study shows that platelet alpha-granule content is unaltered in uncomplicated essential hypertension and suggests that the low platelet serotonin content in hypertensives is mainly due to the inhibition of platelet serotonin uptake.     

Platelet aggregating response to platelet activating factor participates in activation of the 12-lipoxygenase pathway in platelets from rabbits.

BACKGROUND: Many thrombotic angiopathies are known to originate from abnormalities in platelet-blood vessel interaction. The present study focuses on the platelet activating factor (PAF)-induced platelet aggregating mechanism and the arachidonate metabolic pathways activated by it in platelets from a male rabbit. METHODS: Blood vessel contractility was examined using perfused arterial segments dissected from rabbit ear central arteries. Autologous platelet rich plasma (PRP) was infused into the perfusion system. Vascular contractilities were examined during infusion of PRP with a platelet agonist such as collagen or PAF. Platelet aggregation was measured with a platelet aggregometer. RESULTS: Vasocontractile response to noradrenaline (NA) during infusion of PRP with PAF was initially augmented but gradually became attenuated, whereas repetitive vasocontractile responses to NA in the presence of PRP with collagen gradually increased. Platelet aggregation in response to PAF was moderately inhibited by both nordihydroguaiaretic acid (NDGA) and baicalein, 12-lipoxygenase (LOX) inhibitors, was unaffected by indomethacin (IM), a cyclo-oxygenase (COX) inhibitor, and was markedly diminished by EGTA, a calcium chelator. CONCLUSIONS: PAF-induced platelet aggregation may participate in the activation of the 12-LOX pathway rather than the COX pathway in platelets, and is remarkably sensitive to intracellular Ca2+ levels.     

In vivo platelet activation in lower limbs thrombophlebitis. A longitudinal study

Platelet activation in vivo was studied in patients with thrombophlebitis of the lower limbs. The parameters considered were the platelet aggregate ratio (PAR) and the beta-thromboglobulin (beta-tg) level, which were repeatedly evaluated from the disease onset up to 3 months later, during anticoagulating and antiaggregating therapy. A significant decrease of PAR was found, along with a significant rise of the beta-tg level at the onset of the disease, and these values slowly returned to normal on therapy course. The same parameters exceeded the normal range again when the patients arbitrarily suspended any drug assumption. The possible significance and implications of these findings are discussed.     

Tissue factor pathway inhibitor in health and disease

The knowledge of tissue factor pathway inhibitor (TFPI) has greatly expanded during the last few years, and TFPI is now established as a coagulation inhibitor of great importance. Its main role seems to be inhibition of small amounts of tissue factor, which probably is essential for maintaining a normal hemostatic balance. The acceleration of TFPI's inhibitory effect by heparin, the TFPI release caused by heparin injection, and TFPI's heparin affinity may greatly contribute to the anticoagulant properties of the endothelium, and may be particularly important for the outcome of vascular injury. The information provided by one single measurement of plasma TFPI in a patient is difficult to interpret, but serial measurements during the course of a disease may signal the prognosis. Recombinant TFPI has proved effective in the treatment of experimental disseminated intravascular coagulation, sepsis, and thrombosis. Whether TFPI or TFPI derivatives will be as effective in the treatment of patients remains to be determined.     

Tissue factor pathway inhibitor: the carboxy-terminus is required for optimal inhibition of factor Xa.

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type protease inhibitor that binds to and inactivates factor Xa directly, and in a factor Xa-dependent fashion inhibits the factor VIIa/tissue factor catalytic complex. TFPI is a slow, tight-binding, competitive, and reversible inhibitor of factor Xa, in which the formation of an initial encounter complex between TFPI and factor Xa is followed by slow isomerization to a final, tightened complex. Wild-type recombinant TFPI (rTFPI), expressed in mouse C127 cells, separates into two forms on heparin-agarose chromatography that elute at 0.3 mol/L and 0.6 mol/L NaCl. Western blot analysis shows that both forms contain the N-terminus of full-length TFPI, but only rTFPI(0.6) is recognized by an antibody directed against the C-terminus. rTFPI(0.3) and rTFPI(0.6) inhibit factor Xa with 1:1 stoichiometry and inhibit factor VIIa/tissue factor equally in an endpoint-type assay. However, rTFPI(0.6) is a more potent inhibitor than rTFPI(0.3) of coagulation in normal plasma induced by either factor Xa or tissue factor. The initial inhibition of factor Xa (less than 5 seconds) produced by rTFPI(0.6) is several-fold greater than that produced by rTFPI(0.3), presumably reflecting a lower Ki of the immediate encounter complex between factor Xa and TFPI. The differential effect of these forms of TFPI on tissue factor-induced coagulation in normal plasma appears to be directly related to their ability to inhibit factor Xa. To confirm the role of the C-terminal region of TFPI in optimal factor Xa inhibition, a carboxy-terminal mutant of rTFPI, which is truncated after leucine 252 and thus lacks the basic sequence K T K R K R K K Q R V K (residues 254-265), was expressed in C127 cells. This form of rTFPI elutes from heparin-agarose at 0.28 mol/L NaCl and inhibits factor Xa at a rate that is slower than rTFPI(0.3). The Ki(final)s for factor Xa inhibition by rTFPI(0.6), rTFPI(0.3), and rTFPI1-252 are 3.1 +/- 0.6, 19.6 +/- 0.8, and 19.6 +/- 3.0 pmol/L, respectively.     

Genetic variants in platelet factor 4 modulate inflammatory and platelet activation biomarkers

Background- African Americans suffer from higher prevalence and severity of atherosclerosis compared with whites, highlighting racial and ethnic disparities in cardiovascular disease. Previous studies have pointed to the role of vascular inflammation and platelet activation in the formation of atherosclerotic lesions. Methods and Results- We explored the role of genetic variation in 4 chemokine/chemokine receptor genes (CX3CR1, CX3CL1, CXCR3, and PF4) on systemic inflammation and platelet activation serum biomarkers (fractalkine, platelet P-selectin, platelet factor 4 [PF4], and tumor necrosis factor-α). In total, 110 single nucleotide polymorphisms were tested among 1042 African Americans and 763 whites. The strongest association with serum PF4 levels was observed for rs168449, which was significant in both racial groups (P value: African Americans=0.0017, whites=0.014, combined=1.2 × 10(-4)), and remained significant after permutation-based multiple corrections (P(c) value: combined=0.0013). After accounting for the effect of rs168449, we identified another significant single nucleotide polymorphism (rs1435520), suggesting a second independent signal regulating serum PF4 levels (conditional P value: African Americans=0.02, whites=0.02). Together, these single nucleotide polymorphisms explained 0.98% and 1.23% of serum PF4 variance in African Americans and whites, respectively. Additionally, in African Americans, we found an additional PF4 variant (rs8180167), uncorrelated with rs168449 and rs1435520, associated with serum tumor necrosis factor-α levels (P=0.008, P(c)=0.048). Conclusions- Our study highlights the importance of PF4 variants in the regulation of platelet activation (PF4) and systemic inflammation (tumor necrosis factor-α) serum biomarkers.     

Increased thromboxane biosynthesis in patients with polycythemia vera: evidence for aspirin-suppressible platelet activation in vivo.

Increased thromboxane (TX) production and modified aspirin sensitivity has been detected in vitro in platelets isolated from patients with polycythemia vera. To verify the relevance of these capacity-related measurements to the actual rate of TXA2 biosynthesis in vivo and its suppression by oral aspirin, we have investigated the urinary excretion of major enzymatic metabolites of TXB2 in 17 patients with polycythemia vera and 23 gender- and age-matched controls. Urinary 11-dehydro-TXB2 and 2,3-dinor-TXB2 were measured by previously validated radioimmunoassays. In addition, urinary immunoreactive leukotriene (LT) E4 was measured to explore the 5-lipoxygenase pathway of arachidonate metabolism. Polycythemic patients had significantly (P < .001) higher excretion rates of both 11-dehydro-TXB2 (1,033 +/- 1,050 v 117 +/- 45 pmol/mmol creatinine; mean +/- SD) and 2,3-dinor-TXB2 (725 +/- 676 v 82 +/- 43 pmol/mmol creatinine) than controls. In contrast, urinary LTE4 was not significantly different. Enhanced metabolite excretion did not correlate with the platelet count or with the hematocrit value, and was not related to the current treatment or to a clinical history of thrombotic complications. Platelet TX receptor studies did not show any significant changes in the binding characteristics of two different ligands. A platelet-selective regimen of aspirin therapy (50 mg/d for 7 to 14 days) was associated with greater than 80% suppression in metabolite excretion in nine patients. These results are consistent with abnormal stimuli operating in polycythemia vera to induce a selective enhancement in the platelet biosynthesis of TXA2 without changes in receptor binding. This in vivo abnormality in platelet biochemistry can be largely suppressed by low doses of aspirin.     

Myocardial infarct of effort with normal coronaries. Study of in vivo platelet activation

Platelet activation may play a part in causing myocardium infarction with angiographically normal coronary arteries. We investigated this possibility by performing ergometric stress testing in a series of 9 patients (Group A) who had suffered myocardial infarction after a violent effort with angiographically documented coronary insufficiency responsible for a stable effort angina (Group B) and 11 healthy subjects (Group C). Blood samples were taken separately before exercise, at the peak of exercise, and during the recovery period. Platelet morphology, a sensitive indication of the degree of platelet activation, was studied by phase contrast microscopy after immediate fixation of the blood. The percentage of non-discoidal platelets presenting with one or several spicules was measured. At the same time, the plasma concentrations of platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG) were measured. At rest, there was no difference in platelet morphology or specific platelet proteins between the 3 groups. At the peak effort, there was a significant increase of the number of morphologically modified platelets in Groups A and B but not in healthy subjects. This platelet activation could not be linked to the presence of myocardial ischaemia because it was found both in patients with a negative maximal exercise stress test (Group A). Finally, no increase of the plasma concentrations of the platelet protein was observed in any of the groups.     

Interaction of platelets and blood vessels--vascular injuries induced by platelet activation in vivo.

We have found that vascular injuries are induced by intravascular aggregation of platelets activated by arachidonic acid (AA) or ADP. The characteristic findings are the appearance of vacuoles in endothelial cells and eventual deendothelialization. In deendothelialized regions, formation of platelet thrombi was observed. The platelets in the thrombi were stained with 2T60, a monoclonal antibody that recognizes activated platelets. The change was more remarkable in the AA-injected animals because AA has a stronger platelet activating effect and a detergent effect on the endothelium. The ADP-injection experiments clarified the role of platelets in vascular injury. These findings suggest that activated platelets play a role in the genesis of vascular injuries, and that their role is related to thrombus formation.     

Platelet von Willebrand factor: evidence for its involvement in platelet adhesion to collagen

Although it is well established that plasma von Willebrand Factor (vWF) is essential to platelet adhesion to subendothelium at high shear rates, the role of platelet vWF is less clear. We studied the respective role of both plasma and platelet vWF in mediating platelet adhesion to fibrillar collagen in a parallel-plate perfusion chamber. Reconstituted blood containing RBCs, various mixtures of labeled washed platelets and plasma from controls or five patients with severe von Willebrand disease (vWD), was perfused through the chamber for five minutes at a shear rate of 1,600 s-1. Platelet-collagen interactions were estimated by counting the radioactivity in deposited platelets and by quantitative morphometry. When the perfusate consisted of normal platelets suspended in normal plasma, platelet deposition on the collagen was 24.7 +/- 3.6 X 10(6)/cm2 (mean +/- SEM, n = 6). Significantly less deposition (16 +/- 2.3) was observed when vWD platelets were substituted for normal platelets. In mixtures containing vWD plasma, significantly greater deposition (9 +/- 2.2) was obtained with normal than with vWD platelets (1 +/- 0.4) demonstrating a role for platelet vWF in mediating the deposition of platelets on collagen. Morphometric analysis confirmed these data. Our findings indicate that platelet, as well as plasma, vWF mediates platelet-collagen interactions at a high shear rate.