Kininogen (HK)-based thrombin inhibitor selectively and completely blocks thrombin-induced platelet aggregation by irreversible inhibition of platelet calpain which mediates the reaction (IC50= 4.14 µM). This peptide does not inhibit platelet aggregation induced by hemostasis factors other than thrombin.

In 1991, Puri et al., showed that high molecular weight HK (1) inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets; (2) HK is a specific inhibitor of platelet aggregation induced by a- and ?-thrombin; and (3) HK plays a role in modulating platelet aggregation stimulated by a-thrombin in plasma 1. Later in 1993 they showed that the highly conserved sequence Gln-Val-Val-Ala-Gly, present in HK and its evolutionary precursors, the cystatins, is involved in the binding of cysteine proteases and an affinity analog,  called P1, was an irreversible inhibitor of platelet calpain 2.

Structural Characteristics
It is an affinity analog, amino acid sequence is Phe-Gln-Val-Val-Cys(Npys)-Gly-NH2 (Npys, 3-nitro-2-sulfenylpyridine) designated as P1, corresponding to the thiol-protease-binding sequence in HK and containing a ligand, Npys, that can react with the free sulfhydryl group in the active site of calpain 2.

Mode of Action
Thrombin-induced platelet aggregation has been suggested to play an important role in reocclusion following thrombolytic therapy of angioplasty for treatment of myocardial infarction. Puri et al., demonstrated that aggregation of washed platelets by thrombin is accompanied by cleavage of aggregin, a putative ADP receptor, and these events are indirectly mediated by calpain, expressed on the surface of the external membrane. High-molecular-mass HK contains, in its heavy chain, domain 2, which is responsible for its action as a potent inhibitor of platelet calpain. Domain 3 of the heavy chain of HK directly inhibits binding of thrombin to platelets, confounding mechanistic studies using the entire molecule. HK, a protease of 120 kDa, is unsuitable as a potential pharmacological agent. The highly conserved sequence Gln-Val-Val-Ala-Gly, present in HK and its evolutionary precursors, the cystatins, is known to be involved in the binding of cysteine proteases but is, itself, not inhibitory. An affinity analog, P1, was an irreversible inhibitor of platelet calpain. P1 selectively inhibited thrombin-induced aggregation of washed platelets and platelets in plasma, but did not inhibit the aggregatory effects of other platelet agonists. P1 did not inhibit the amidolytic activity and coagulant activity of thrombin. Unlike HK, P1 did not inhibit binding of thrombin to washed platelets. P1 did not inhibit thrombin-induced platelet-shape change. P1 neither raised intracellular levels of cAMP nor did it interfere with the ability of thrombin to antagonize the rise in intracellular levels of cAMP induced by iloprost, an analog of prostaglandin I2 2.


Functions on endothelium,
Bradykinin (BK) is a vasoactive peptide that is released from the plasma kininogens. It has been described as having multiple physiological functions on endothelium. BK in HK participates in the binding of its parent molecules, kininogens, to endothelium. It is a potent stimulator of prostacyclin formation 3, 4.

Thrombin inhibitor, HK can also function as an indirect -thrombin inhibitor by blocking -thrombin–induced, membrane-expressed platelet calpain from contributing to platelet aggregation. BK and its analogues inhibit -thrombin–induced platelet activation suggests a third mechanism by which HK and LK (low-molecular-weight kininogen) are antithrombins. Each of these antithrombotic activities of kininogens could be contributing to the constituent anticoagulant nature of the intravascular compartment. In fact, the presence of HK in human plasma requires a 2.5-fold increase in the concentration of -thrombin to induce platelet aggregation in platelet-rich plasma 5, 2.

Platelet aggregation, Initial studies with platelet aggregation and secretion suggest that these peptides may be inhibiting platelet activation via more than one mechanism. The better inhibition of platelet aggregation compared with platelet secretion as measured through [14C]-5-hydroxytryptamine secretion suggested that these peptides may be interfering with fibrinogen binding to platelets 6.The design and synthesis of P1 has lead to the development of a new class of inhibitors that selectively block thrombin-induced platelet aggregation while sparing other functions of this pathophysiological protease and without inhibiting the action of other platelet agonists.


1.     Puri RN, Zhou F, Hu C-J, Colman RF, Colman RW (1991). High molecular weight kininogen inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets. Blood, 77:500-507.

2.     Puri RN, Matsueda R, Umeyama H , Badford HN, Colman RW (1993). Modulation of thrombin-induced platelet aggregation by inhibition of calpain by a synthetic peptide derived from the thiol-protease inhibitory sequence of kininogens and S-(3-nitro-2-pyridinesulfenyl)-cysteine. European Journal of Biochemistry, 214(1):233-241.

3.     Hong SL (1980). Effect of bradykinin and thrombin on prostacyclin synthesis in endothelial cells from calf and pig aorta and human umbilical cord vein. Thromb Res., 18:787-796.

4.     Crutchley DJ, Ryan JW, Ryan US, Fisher GH (1983). Bradykinin-induced release of prostacyclin and thromboxanes from bovine pulmonary artery endothelial cells. Biochim Biophys Acta., 751:99-107.

5.     Schmaier AH, Bradford HN, Lundberg D, Farber A, Colman RW (1990). Membrane expression of platelet calpain. Blood, 75:1273-1281.

6.     Nakashima M, Mombouli JV, Taylor AA, Vanhoutte PM (1993). Endothelium-dependent hyperpolarization caused by bradykinin in human coronary arteries. J Clin Invest., 92:2867-2871.

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