All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.
One of the major physiological roles of the endothelium is to preserve the integrity of the vasculature by its permeability barrier properties, and to provide a non-thrombogenic surface. Therefore, the endothelial cell surface serves as a prime regulatory site of coagulation responses. Under normal circumstances, an injury to vascular endothelial cells lining a blood vessel triggers a haemostatic response through a sequence of events commonly referred to as the “coagulation cascade”. The cascade culminates in the conversion of soluble fibrinogen to insoluble fibrin which, together with platelets, forms a localized clot or thrombus which prevents extravasation of blood components. Wound healing can then occur followed by clot dissolution and restoration of blood vessel integrity and flow.
The events which occur between injury and clot formation are carefully regulated and linked series of reactions, involving number of plasma coagulation proteins in inactive proenzyme forms and cofactors circulate in the blood. Active enzyme complexes are assembled at an injury site and are sequentially activated to serine proteases, with each successive serine protease catalyzing the subsequent proenzyme to protease activation. This enzymatic cascade results in each step magnifying the effect of the succeeding step. In brief, the blood coagulation cascade is usually initiated as soon as the cell surface glycoprotein tissue factor (TF) comes into contact with circulating activated factor VII (VIIa), resulting in the formation of TF-FVIIa complex. The TF-VIIa complex activates X to factor Xa. Subsequently, factor Xa catalyzes the conversion of prothrombin to thrombin, thereby leading to fibrin formation, platelet activation, and, ultimately, generation of a thrombus. Tissue factor pathway inhibitor (TFPI) is a potent direct inhibitor of factor Xa, and in a factor Xa dependent fashion, produces inhibition of the factor VIIa-TF complex.
While efficient clotting limits the loss of blood at an injury site, inappropriate formation of thrombi in veins or arteries is a common cause of disability and death. Abnormal clotting activity can result in and/or from pathologies or treatments such as myocardial infarction, unstable angina, atrial fibrillation, stroke, renal damage, percutaneous translumenal coronary angioplasty, disseminated intravascular coagulation, sepsis, gestational vascular complications, pulmonary embolism and deep vein thrombosis. The formation of clots on foreign surfaces of artificial organs, shunts and prostheses such as artificial heart valves is also problematic.
Approved anticoagulant agents currently used in treatment of these pathologies and other thrombotic and embolic disorders include the sulfated heteropolysaccharides heparin and low molecular weight heparin (LMWH). These agents are administered parenteral and can cause rapid and complete inhibition of clotting.
Heparanase is an endo-β-D-glucuronidase capable of cleaving heparan sulfate (HS) side chains at a limited number of sites, yielding HS fragments of still appreciable size (˜5-7 kDa) [Freeman, C. and Parish, C. R. Biochem. J. 330:1341-1350 (1998); Pikas, D. S. et al. J. Biol. Chem. 273:18770-18777 (1998)]. Expression of heparanase is restricted primarily to the placenta, platelets, keratinocytes, and activated cells of the immune system, with little or no expression in connective tissue cells and most normal epithelia [Parish, C. R. et al. Biochem. Biophys. Acta 1471:M99-108 (2001); Vlodaysky, I. and Friedmann, Y. J. Clin. Invest. 108:341-347 (2001)]. During embryogenesis, the enzyme is preferentially expressed in cells of the developing vascular and nervous systems. Heparanase activity was implicated in tumor growth, neovascularization, inflammation and autoimmunity [Parish (2001) ibid.; Vlodaysky (2001) ibid.; Dempsey, L. A. et al. Trends Biochem. Sci. 25:349-351 (2000)]. A single human heparanase cDNA sequence was independently reported by several groups [Hulett, M. D. et al. Nat. Med. 5:803-809 (1999); Kussie, P. H. et al. Biochem. Biophys. Res. Commun. 261:183-187 (1999); Toyoshima, M. and Nakajima, M. J. Biol. Chem. 274:24153-24160 (1999); Vlodaysky, I. et al. Nat. Med. 5:793-802 (1999)]. Thus, unlike the large number of proteases that can degrade polypeptides in the extra-cellular matrix (ECM), one major heparanase appears to be used by cells to degrade the HS side chains of HS proteolycans.
Heparanase is synthesized as a latent 65 kDa precursor whose activation involves proteolytic cleavage at two potential sites located at the N-terminal region of the molecule (Glu109-Ser110 and Gln157-lys158), resulting in the formation of two protein subunits, 8 and 50 kDa polypeptides, that heterodimerize and form the active heparanase enzyme [McKenzie, E. et al. Biochemical J. 373: 423-35 (2003); Levy-Adam F. et al. Biochem. Biophy. Res. Commun. 308: 885-91 (2003)]. One of the prime physiological sources for heparanase are platelets [Hulett (1999) ibid.; Freeman and Parish (1998) ibid.]. The 50 and 8 kDa heparanase polypeptides were biochemically purified from platelets, which also contain significant amounts of the 65 kDa proenzyme [Hulett (1999) ibid; Freeman (1998) ibid.]. Moreover, the heparanase gene was previously cloned from human platelets [Hulett (1999) ibid.]. Heparanase released by activated platelets or platelet-derived microparticles, is biologically active, stimulates angiogenesis and modulates endothelial cell activities [Brill, A. et al. Cardiovasc. Res. 63:226-235 (2004); Myler, H. A. and West, J. L. J. Biochem. 131:913-922 (2002)].
It has been previously shown by applying heparanase which lacks enzymatic activity that heparanase exerts also non-enzymatic activities, independent of its involvement in ECM degradation and alterations in the extracellular microenvironment associated with angiogenesis, cell survival, and migration [Goldshmidt, O. Faseb. J. 17:1015-1025 (2003); Gingis-Velitski, S. et al. J. Biol. Chem. 279:23536-23541 (2004); Zetser, A. et al. Cancer Res. 66:1455-1463 (2006)].
The inventors have recently demonstrated that heparanase is also involved in the hemostatic system. Heparanase was shown to up-regulate TF expression [Nadir, Y. J. et al. Thromb. Haemost. 4:2443-2451 (2006)] and interact with TFPI on the cell surface, leading to dissociation of TFPI from the cell membrane and increased cell surface coagulation activity [Nadir, Y. et al. Thromb. Haemost. 99:133-141 (2008)].
When the present inventors further examined the role of heparanase in coagulation modulation, they surprisingly and unexpectedly found, as shown by the present invention, that heparanase is directly involved in the activation of the coagulation system by enhancing factor Xa production in the presence of TF/VIIa complex.
More particularly, addition of heparanase to TF and factor VIIa resulted in three to four fold increase in activation of the coagulation cascade as depicted by increased factor Xa and thrombin production. When heparanase was added to pooled normal plasma a seven to eight fold increase in Xa level was observed. Subsequently, the inventors looked for clinical data supporting the existence of this innovative role of heparanase. Plasma samples of thirty five patients with acute leukemia at presentation, and twenty healthy controls were studied for heparanase and factor Xa levels using ELISA and chromogenic assay, respectively. A strong positive correlation was found between plasma heparanase and Xa levels (r=0.735, p=0.001). In addition, unfractionated-heparin and an anti-Xa derivative abolished the effect of heparanase, while, TFPI and TFPI-2 only attenuated the procoagulant effect. Finally, co-immunoprecipitation and far-western analyses demonstrated that heparanase directly interacts with TF. Altogether, the results presented herein support the notion that heparanase is a modulator of blood homeostasis, and suggest a novel mechanism by which heparanase is involved in direct activations of the coagulation cascade by direct interaction with TF.
For the purpose of better assessing the procoagulant role of heparanase in various medical conditions, the inventors have developed an original assay to evaluate heparanase procoagulant activity in biological samples. Heparanase procoagulant activity was preliminary studied using purified proteins of heparanase, TF, factor VIIa and factor X. The methodology was verified in fifty five plasma samples and compared to heparanase and tissue factor pathway inhibitor (TFPI) levels by ELISA and factor Xa, thrombin levels and antithrombin activity by chromogenic substrates. Thirty five samples were of third-trimester pregnant women (weeks 36-41) who were in labor or came for appointed elective cesarean section and 20 control samples were of non-pregnant healthy women. The heparanase procoagulant activity assay was shown to differentiate heparanase procoagulant effect from TF activity, in purified proteins model. Heparanase procoagulant activity was significantly higher in the plasma of pregnant women compared to non-pregnant (p<0.005). Heparanase relative contribution to the TF heparanase complex activity was significantly higher in the plasma of pregnant women compared to non-pregnant (29% increase, p<0.0001). Differences in heparanase procoagulant activity were more prominent than changes in heparanase levels by ELISA, TF activity, factor Xa, thrombin and free TFPI levels. The results presented therein demonstrate that heparanase procoagulant activity can be determined by the assay and the methodology of the present invention. In this particular case, the assay of the invention revealed a significant contribution of heparanase to the procoagulant state in late third-trimester pregnancy and at delivery.
The methodology and the assay developed in the present invention were then applied to the field of orthopedic hip and knee surgeries which are followed by a hypercoagulable state. Heparanase level and procoagulant activity in patients undergoing orthopedic surgery were assessed. The study group included fifty orthopedic patients. Thirty one patients underwent hip surgery and nineteen had knee operation. Fifteen individuals suffered from traumatic hip fractures and thirty five had osteoarthrosis of hip or knee joints. All patients received prophylactic dose of enoxaparin (a low molecular weight heparin) starting 6-8 hours post operation and lasting for five weeks. Plasma samples were drawn preoperatively and at 1 hour, 1 week and 1 month post operation. Samples were tested for heparanase levels by ELISA and TF/heparanase complex activity, TF activity, heparanase procoagulant activity (with the assay of the present invention), factor Xa and thrombin levels using chromogenic substrates. Heparanase levels were significantly higher 1 hour and 1 week post operatively compared to preoperative levels (p<0.05, p<0.005, respectively). The most dramatic changes were observed in heparanase procoagulant activity reaching a 2 fold increase 1 week postoperatively and 1.7 fold increase 1 month after surgery (p<0.0001, p<0.0001, respectively). Levels of factor Xa and thrombin did not significantly change. In conclusion, the inventors demonstrated that heparanase is involved in coagulation activation of orthopedic surgery patients. Heparanase procoagulant activity is highest 1 week postoperatively and remains high 1 month after operation. In view of the above, considering extending prophylactic anticoagulant therapy or evaluating heparanase procoagulant activity may potentially prevent late thrombotic events.
It is therefore one object of the invention to provide a composition comprising a combination of heparanase or any fragments, derivative or peptides thereof and TF or any fragments, derivatives or peptides thereof, specifically for use in modulation of coagulation processes.
In yet another object, the invention provides a method for the activation of a pro-coagulation process in a subject in need thereof, using the combined heparanase-TF compositions of the invention. These methods are specifically applicable in the treatment, amelioration and prevention of coagulation-related pathologic conditions
In yet another object, the invention provides the use of the novel heparanase-TF interaction as a target in a screening method for identification of coagulation-modulating compounds. Such compounds modulate the heparanase-TF mediated coagulation activity in a subject suffering of a coagulation-related disorder.
In yet another object, the invention provides a method for determining heparanase procoagulant activity in a biological sample of a mammalian subject.
In yet another object, the invention provides a diagnostic method for the detection and/or monitoring of a coagulation-related pathologic disorder in a mammalian subject.
In yet another object, the invention provides a diagnostic kit for the detection and/or monitoring of a coagulation-related pathologic condition in a mammalian subject.
These and other objects of the invention will become apparent as the description proceeds.