Kininogens Are Antithrombotic Proteins In Vivo

Abstract

Abstract-Kininogens have recently been shown to possess antiadhesive, anticoagulant, and profibrinolytic properties and can inhibit platelet activation at low thrombin concentrations. To test whether kininogens have antithrombotic properties in vivo, we devised a model of limited arterial injury confined to removal of the endothelium. Brown-Norway Katholiek strain rats with an absence of low-and high-molecular-weight kininogen due to a single point mutation, A163T, were compared in the thrombosis model to the wild-type animals, which were otherwise genetically identical. Despite an equivalent vascular injury, the mean time (ϮSEM) for a 90% decrease in flow measured by laser Doppler was 38.4Ϯ17 minutes in the kininogen-deficient rats compared with 194Ϯ29 minutes in the wild-type animals (PϽ0.002). Key Words: arterial thrombosis Ⅲ kininogens Ⅲ antithrombotic Ⅲ fibrinolysis Ⅲ rats K ininogens are plasma proteins first recognized as the precursors of a peptide, bradykinin, which was liberated by plasma kallikrein. 1 Bradykinin can reproduce many inflammatory changes, including edema, pain, vasodilation, and increased vascular permeability. A deficiency of plasma high-(HK) and low-(LK) molecular-weight kininogens 2 in humans does not lead to a hemorrhagic disorder, despite a marked prolongation of the activated partial thromboplastin time. In fact, cloning 3 and delineation of the structurefunction relationships of kininogen have revealed new properties of this protein, including cysteine protease inhibition, 4 modulation of thrombin-induced platelet aggregation, 5 antiadhesive properties, 6 and profibrinolytic potential. 7 HK and LK have been divided into domains D1, D2, and D3, which compose the common heavy-chain N-terminal to domain D4, which contains the bradykinin sequence. LK contains a small domain, D5 L , whereas HK contains the large domains D5 H and D6, which compose the light chains. 3 The ability of D2 of the kininogens to inhibit platelet calpain 4 has been linked to an inhibition of thrombin-induced platelet aggregation, 8 whereas inhibition of the binding of thrombin to platelets by D3 of kininogens has led to a 10-fold shift in the doseresponse curve of thrombin-induced platelet activation. 9 HK can compete for fibrinogen binding to neutrophils 10 by competing for a site on the integrin Mac-1 11 and thus, limit the adhesion of neutrophils to artificial and biological surfaces. Peptides from D6 of HK can downregulate urokinasedependent plasminogen activation on human endothelial cells 7 by inhibiting the binding of prekallikrein to HK.All of these studies suggest that HK should serve as an antithrombotic protein. Conversely, individuals deficient in HK should manifest a prothrombotic state. Unfortunately, HK deficiency is a rare condition, and only a very small number of individuals have been observed in a serial fashion. Therefore, to test this hypothesis, we decided to use a well-defined animal model. Fortunately, a natural "knockout" exists in the Katholiek strain of the Brown-Norway rat. These rat...

A Novel Antithrombotic Role for High Molecular Weight Kininogen as Inhibitor of Plasminogen Activator Inhibitor-1 Function

“…Conformational differences between HK and HKa, especially the more prominent exposure of the described region in HKa compared with HK (49), may explain the significantly higher efficiency of HKa over HK for inhibition of PAI-1 activity. Taken together, our data emphasize that the inhibition of PAI-1 function by the sequence Gly 486 -Lys 502 derived from domain 5 of HK provides a plausible explanation for the previously described antithrombotic role of kininogen (30,34). Based on these observations, potential low molecular weight PAI-1 inhibitors could be designed for antithrombotic therapeutic interventions.…”

“…(iv) The binding of HKa to the urokinase receptor (66) on vascular cell surfaces may approximate kallikrein and prourokinase and thereby potentiate plasmin formation. (v) HK-deficient rats are presented with a prothrombotic phenotype (30). Together, various domains/portions of HK can potentially contribute to the antithrombotic role of this plasma protein, and in particular the multifunctional domain 5 may be used as a leading substance for therapeutic interventions in vascular pathologies.…”

“…We have demonstrated previously that HKa and PAI-1 compete for proximal binding sites within the N-terminal "somatomedin B" domain of VN (24). Experimental evidence from in vitro and in vivo studies renders HK antirather than prothrombotic; a prothrombotic phenotype was reported for kininogen-deficient rats (30), and patients deficient in kininogen or other proteins of the contact phase system are at increased risk for thrombosis (31)(32)(33)(34). In particular, HK/HKa may regulate pericellular plasmin generation by modulating plasma kallikrein-dependent formation of urokinase plasminogen activator (uPA) (35), a reaction dependent on the binding of plasma prekallikrein to HK domain 6 (36).…”

Kininogen deficiency protects from ischemic neurodegeneration in mice by reducing thrombosis, blood-brain barrier damage, and inflammation

“…First, several reports have suggested that KNG exerts antithrombotic effects. [26][27][28] However, our studies clearly found that the absence of KNG protects from thrombosis in the tMCAO model. The exact reasons for these divergent findings are unclear so far.…”

Antithrombotic activity of kininogen is mediated by inhibitory effects of domain 3 during arterial injury in vivo

“…However, the Brown-Norway-Katholiek (B/N/ Ka) rat strain has severe deficiency of both plasma kininogens (12) due to a single point mutation, Ala163 to Thr, which results in defective secretion from the liver. We have shown (7) that occlusive thrombosis resulting from deendothelization of the rat aorta is sixfold more rapid in B/N/Ka rats with kininogen deficiency than in Kitasato rats (B/N/Ki) with normal kininogen, indicating that kininogen is antithrombotic in vivo. However, two limitations of that study should be acknowledged.…”