Platelet-rich Clot Lysis Research Articles

Integration of VEK-30 peptide enhances fibrinolytic properties of staphylokinase.

Staphylokinase (SAK), a 136 amino acid bacterial protein with profibrinolytic properties, has emerged as an important thrombolytic agent because of its fibrin specificity and reduced inhibition by α-2 antiplasmin. In an attempt to enhance the clot dissolution ability of SAK, a 30 amino acid peptide (VEK-30) derived from a plasminogen (Pg) binding protein (PAM), was fused at the C-terminal end of SAK with a RGD (Arg-Gly-Asp) linker. The chimeric protein, SAKVEK, was expressed in E. coli and purified as a soluble protein. Pg activation by equimolar complexes of SAKVEK and SAK with plasmin revealed that the fusion of VEK-30 peptide has significantly enhanced the catalytic activity of SAK. The kinetic constant, kcat /Km , of SAKVEK for the substrate Pg appeared 2.7 times higher than that of SAK and the time required for the fibrin and platelet rich clot lysis was shortened by 30% and 50%, respectively. The binary activator complex of SAKVEK with plasmin gets inhibited by α2- antiplasmin but remains protected in the presence of fibrin, very similar to SAK. Thus, the present study suggests that SAKVEK is more potent and effective as a thrombolytic agent due to its higher catalytic activity for Pg activation in a fibrin-specific manner and its ability to clear platelet-rich plasma clot faster than SAK.

Designing a multifunctional staphylokinase variant (SAK-2RGD-TTI) with appropriate thrombolytic activity in vitro.

Thrombin, platelets, and plasmin are three key factors involved in hemostasis and thrombolysis. Thrombolytic therapy with clinically approved drugs is often followed by recurrent thrombosis caused by thrombin-induced platelet aggregation from the clot debris. In order to minimize these problems, new constructs were designed for the expression of recombinant staphylokinase (rSAK) and also a fusion protein composed of staphylokinase, 20 amino acids containing 2 RGD followed by tsetse thrombin Inhibitor (SAK-2RGD-TTI) in Pichia pastoris. Modeling the tertiary structure of SAK-2RGD-TTI showed that the linker containing RGD and TTI did not interfere with proper folding of SAK. In laboratory testing, the purified SAK-2RGD-TTI (420μg/mL) dissolved an average of 45% of the blood clot. The activity of the SAK-2RGD-TTI was also confirmed in various tests including human plasminogen activation assay, fibrin clot lysis assay, well diffusion method, activated partial thromboplastin time and platelet rich clot lysis assay. Our findings suggest that SAK-2RGD-TTI has improved therapeutic properties preventing reocclussion. It further confirms that it is practicable to assemble and produce a hybrid multifunctional protein that targets hemostatic process at various stages.

Peroxynitrite may affect fibrinolysis via the reduction of platelet-related fibrinolysis resistance and alteration of clot structure

We tested the hypothesis that in vitro peroxynitrite (ONOO−, a product of activated inflammatory cells) may affect fibrinolysis in human blood through the reduction of platelet-related fibrinolysis resistance. It was found that ONOO− (25–300µM) accelerated lysis of platelet-fibrin clots (in PRP) dose-dependently, whereas fibrinolysis of platelet-free clots was slightly inhibited by ≥1000µM stressor. Concentrations of ONOO− affecting the lysis of platelet-rich clots, inhibited clot retraction (CR) in a dose-dependent manner. Thromboelastometry (ROTEM) measurements performed in PRP showed that treatment with ONOO− (threshold conc. 100µM) prolongs clotting time, and reduces alpha angle, and clot formation velocity parameters indicating for reduced thrombin formation rate. In PRP, ONOO− (threshold conc. 100µM) reduced the collagen-evoked exposure of phosphatidylserine (PS) on platelets’ plasma membrane, the shedding of platelet-derived microparticles (PMP), and inhibited platelet-dependent thrombin generation (measured in artificial system), dose-dependently. As judged by confocal microscopy, similar ONOO− concentrations altered the architecture of clots formed in collagen-treated PRP. Clots formed in the presence of ONOO− were less dense and were composed of thicker fibers, which make them more susceptible to lysis. In platelet-depleted plasma, ONOO− (up to milimolar concentration) did not alter clot structure. Blockage of PS exposed on platelets resulted in an alteration of clot architecture toward more prone to lysis. ONOO−, at lysis-affecting concentrations, inhibited the collagen-evoked secretion of fibrinolytic inhibitors from platelets. We conclude that physiologically relevant ONOO− concentrations may accelerate the lysis of platelet-fibrin clots predominantly via downregulation of platelet-related mechanisms including: platelet secretion, clot retraction, platelet procoagulant response, and the alteration in clot architecture associated with it.

In VitroCharacterization of a Multifunctional Staphylokinase Variant with Reduced Reocclusion, Produced from Salt InducibleE. coliGJ1158

The thrombolytic therapy with clinically approved drugs often ensues with recurrent thrombosis caused by thrombin-induced platelet aggregation from the clot debris. In order to minimize these problems, a staphylokinase (SAK)-based bacterial friendly multifunctional recombinant protein SRH (staphylokinase (SAK) linked with tripeptide RGD and dodecapeptide Hirulog (SRH)) was constructed to have Hirulog as an antithrombin agent and RGD (Arg-Gly-Asp) as an antiplatelet agent in the present study. This multifunctional fusion protein SRH was expressed in osmotically inducible E. coli GJ1158 as soluble form and purified with a yield of 0.27 g/L and functionally characterized in vitro. SRH retained the fibrinolytic activity and plasminogen activation rate comparable to the parental counterpart SAK. The antithrombin activity of SRH was significantly higher than SAK. The platelet rich clot lysis assay indicated that SRH had enhanced platelet binding activity and T 50% and C50 of SRH were significantly lower than that of SAK. Furthermore, SRH inhibited the ADP-induced platelet aggregation in dose-dependent manner while SAK had no significant effect on platelet aggregation. Thus, the current study suggests that the SAK variant produced from osmotically inducible GJ1158 is more potent thrombolytic agent with antithrombin and antiplatelet aggregation activities for reduction of reocclusion in thrombolytic therapy.

Synergistic Effects of Bifunctional Antibodies Against beta3 Integrin on Dissolution of Platelet Thrombus,

Abstract 3350HIV-ITP patients have a unique Ab against platelet GPIIIa49-66 which induces oxidative platelet fragmentation in the absence of complement (Cell 106: 551, 2001; JCI 113: 973, 2004). Using a phage display single-chain antibody (scFv) library, we developed a novel human monoclonal scFv Ab against GPIIIa49-66 (named A11), which act similarly to the parental Ab (JBC 283: 3224, 2008). We then produced a bifunctional GPIIIa49-66 agent (named SLK), that targets newly deposited fibrin strands within and surrounding the platelet thrombus and has reduced effects on non-activated circulating platelets (Blood 116: 2336, 2010). In this study, we produced another bifunctional GPIIIa49-66 agent (named APAC), which homes to activated platelets. Like SLK, APAC destroys platelet aggregates ex vivo in an identical fashion with ∼85% destruction of platelet aggregates at 2 hrs. Platelet aggregate dissolution with a combination of SLK and APAC was ∼2 fold greater than either agent alone at 0.025 μM. Platelet-rich clot lysis experiments demonstrated the time required for 50% platelet-rich fibrin clot lysis (T50%) by APAC (95±6.1 min) was significantly longer than that by APAC+SLK (65±7.6 min) at a final concentration of 0.025 μM (APAC+SLK vs APAC, p<0.01). In comparison with APAC alone, the T50% of APAC+SLK was shortened by 1.56, 1.67 and 2.1 fold at the concentrations of 0.025, 0.5 and 0.1μM, respectively. Thus these low concentrations of a combination of both agents are likely to be more effective and less toxic when used therapeutically in vivo. Disclosures:No relevant conflicts of interest to declare.

Dissolution of pre-existing platelet thrombus by synergistic administration of low concentrations of bifunctional antibodies against β3 integrin.

Most antithrombotic approaches target prevention rather than the more clinically relevant issue of resolution of an existing thrombus. In this study, we describe a novel and effective therapeutic strategy for ex vivo clearance of pre-existing platelet thrombus by the combination of two bifunctional platelet GPIIIa49-66 ligands that target different parts of the arterial thrombus. We produced an additional GPIIIa49-66 agent (named APAC), which homes to activated platelets. Like our previously described SLK (which targets newly deposited fibrin strands surrounding the platelet thrombus), APAC destroys platelet aggregates ex vivo in an identical fashion with 85% destruction of platelet aggregates at 2 hours. The combined application of APAC and SLK demonstrated a ∼2 fold greater platelet thrombus dissolution than either agent alone at a low concentration (0.025 µM). Platelet-rich clot lysis experiments demonstrated the time required for 50% platelet-rich fibrin clot lysis (T50%) by APAC (95±6.1 min) or SLK (145±7.1 min) was much longer than that by combined APAC+SLK (65±7.6 min) at the final concentration of 0.025 µM (APAC+SLK vs APAC, p<0.05; APAC+SLK vs SLK, p<0.01). Thus these low concentrations of a combination of both agents are likely to be more effective and less toxic when used therapeutically in vivo.

Fibrinolytic and Antiplatelet Aggregation Properties of a RecombinantCheonggukjangKinase

This study characterized the efficacy of recombinant Cheonggukjang kinase (CGK) 3-5-rich fraction as a thrombolytic agent, which we overexpressed in Bacillus licheniformis ATCC10716, a strain normally lacking fibrinolytic activity. We found that CGK3-5 is a plasmin-like protease that directly degrades fibrin clots and does not activate plasminogen during fibrin clot lysis and platelet-rich clot lysis assays. We also confirmed antiplatelet and antithrombotic activity by CGK3-5-rich fraction both in vitro and in vivo. CGK3-5-rich fraction inhibited collagen-induced platelet aggregation in platelet-rich plasma in a concentration-dependent manner. The concentration of 1.5 mg/mL CGK3-5-rich fraction completely inhibited collagen-induced platelet aggregation. Furthermore, injection of CGK3-5-rich fraction into tail veins dose-dependently protected mice from death by pulmonary embolism induced by collagen and epinephrine. The survival rates were 30%, 70%, and 100%, respectively, with doses of 130 mg/kg, 260 mg/kg, and 520 mg/kg. These findings suggest that CGK3-5 holds promise as a treatment to mitigate the potentially effects of stroke and heart failure.

Thrombolytic activity of Cheonggukjang kinase on the recovery of brain damage in cerebral embolic stroke model

Stroke has become the secondary leading cause of death and the first cause of major adult disability in the world. Cheonggukjang kinase (CGK), referring to the fibrinolytic enzyme that catalyzes the clot dissolution, was prepared from the culture of pHY3‐5 transformed Bacillus subtilis ISW 1214. Fibrinolytic activity of CGK was confirmed by fibrin clot lysis assay and platelet rich clot lysis assay. 40 hr cultured CGK has the highest plasmin‐like protease activity (44.6±1.50 mU/100 μg CGK) compared with other CGKs (24h, 14.0±2.07; 60h, 12.6±0.85; 80h, 16.3±1.36 mU/100 μg CGK). The effect of doses of CGK (0.1–1 U plasmin) on cerebral embolic stroke, injected intravenously for 10 min, 1 hr after middle cerebral artery occlusion (MCAO) showed that all CGK treated groups significantly reduced the infarct volume in a dose‐dependent manner compared to the control group, However, hemorrhage was identified in the coronal brain sections in rats injected 2 U plasmin‐like activity/kg, rt‐PA at 10 mg/kg and CGK at 1 U plasmin‐like activity/kg on cerebral embolic stroke showed similar efficacy. Furthermore, treatment with CGK also improved functional recovery as measured by neurological deficits, Taken together, these findings suggest that CGK may have a great promise in the treatment of stroke.

Functional properties of a novel mutant of staphylokinase with platelet-targeted fibrinolysis and antiplatelet aggregation activities

The present study was performed to characterize the functional properties of RGD-SAK, a novel mutant of staphylokinase (SAK). Biochemical analysis indicated that RGD-SAK maintained the similar structure and the fibrinolytic function of SAK. Measurement of platelet binding activity in vitro demonstrated that RGD-SAK had a much higher affinity with platelets than SAK. In vitro platelet-rich clot lysis assay demonstrated that the engineered mutant outperformed the non-manipulated SAK. The time required for 50% platelet-rich clot lysis was reduced significantly across different concentrations of RGD-SAK comparing with SAK. Meanwhile, RGD-SAK was found to inhibit ADP-induced platelet aggregation in a concentration-dependent manner while SAK had negligible effect on platelet aggregation. In concordance, further study in a porcine coronary balloon injury model demonstrated the efficacy of RGD-SAK for the lysis of platelet-rich coronary blood clots and for the prevention of reocclusion after thrombolysis. These results suggested that RGD-SAK may serve as a potential thrombolytic agent with platelet-targeted fibrinolysis and antiplatelet aggregation activities.

Characterization of a novel bifunctional mutant of staphylokinase with platelet-targeted thrombolysis and antiplatelet aggregation activities

BackgroundAlthough staphylokianse (SAK) is among the most promising blood dissolving agents, it is far from ideal. It is interesting to hypothesize that the clot lysis efficacy of SAK can be enhanced with direct active platelet binding ability, and at the same time the rethrombosis complication after successful recanalization can be minimized with an antiplatelet aggregation activity. The present study was performed to characterize the functional properties of RGD-SAK, a novel mutant of staphylokinase (SAK).ResultsBy using site-directed mutagenesis, an Arg-Gly-Asp (RGD) motif was engineered in the staphylokinase (SAK). This mutant of SAK designated RGD-SAK was expressed, purified and characterized. Biochemical analysis indicated that RGD-SAK maintained the similar structure and the fibrinolytic function of SAK. Measurement of platelet binding activity in vitro demonstrated that RGD-SAK had a much higher affinity with platelets than SAK. In vitro platelet-rich clot lysis assay demonstrated that the engineered mutant outperformed the non-manipulated SAK. The time required for 50% platelet-rich clot lysis and the concentration required to obtain 50% clot lysis (C50) were reduced significantly across different concentrations of RGD-SAK comparing with SAK. Meanwhile, RGD-SAK was found to inhibit ADP-induced platelet aggregation in a concentration-dependent manner while SAK had negligible effect on platelet aggregation.ConclusionRGD-SAK possessed the bifunction to target platelet-rich clots and to block platelets aggregation, and thus may serve as a more potential thrombolytic agent with platelet-targeted thrombolytic and antiplatelet aggregation activities in compared with SAK.

Plasminogen activator inhibitor 1 (PAI-1): in vitro activities and clinical relevance.

Plasminogen activator inhibitor 1 (PAI-1) is the main physiological inhibitor of tissue-type and urokinase-type plasminogen activators (t-PA, u-PA). Endothelial or platelet-derived PAI-1 is normally complexed to vitronectin, which prolongs its half-life and makes it the most efficient inhibitor of activated protein C (APC) and thrombin, in the absence of heparin. These target proteinases share a positively charged surface loop (VR-1 or 37-loop), essential for rapid inactivation of the target enzymes. PAI-1 competes with thrombomodulin for binding this surface-loop in thrombin, which, in combination with APC inhibition, makes it a local procoagulant. Vitronectin enhances PAI-1 fibrin-binding, which prevents premature fibrinolysis but also compromises arterial, platelet-rich clot-lysis during pharmacological thrombolysis. Matrix-form vitronectin binds PAI-1 to regulate both u-PA induced pericellular proteolysis and integrin-substratum interactions, thereby influencing cell migration in angiogenesis, cancer and atherosclerosis. PAI-1 has not been established as an independent risk factor for various chronic cardiovascular diseases, caused by the many sites and pathways that integrate into PAI1 plasma levels. A vigorously increased PAI-1 level, however, results in local fibrin deposition in many inflammatory reactions and it strongly predicts the clinical outcome of sepsis by aggravating disseminated intravascular coagulation. Through its interactions with t-PA, thrombin, APC and thrombomodulin, elevated PAI-1 induces a local procoagulant state at the endothelial cell surface, which is aggravated in combination with other procoagulants like factor V-Leiden. The integrity of blood flow is under the continuous surveillance of the coagulation system, ensuring a rapid formation of fibrinstabilized blood clots at sites of injury to the vessel wall, thereby preventing excessive loss of blood from the circulation. Fibrin is formed from fibrinogen through limited proteolysis by thrombin. These thrombin-generated blood clots, however, need to be disposed of as soon as the damaged vessel wall has been repaired. Clot lysis is carried out by the serine protease plasmin, which cleaves multiple peptide bonds in the fibrin matrix, resulting in soluble fibrin degradation products thereby purging the supporting fibrin network of the clot, a process referred to as fibrinolysis. The activity of both coagulation and fibrinolysis needs to be finely tuned to prevent both premature or delayed clot formation and lysis. When bound to the endothelial surface protein thrombomodulin, thrombin also has anticoagulant activities through the protein C pathway, resulting in negative feedback inhibition (Esmon, 2000). Therefore both activation and subsequent inhibition of both thrombin and plasmin are tightly regulated in complex feedback regulatory loops, comprised of multiple zymogens, co-activators and inhibitors (Esmon, 2000). Active plasmin is formed from its inactive zymogen, plasminogen, by the specific proteolytic action of tissue-type plasminogen activator (t-PA). Plasminogen can also be activated by the urokinase-type plasminogen activator (uPA), which, in contrast to t-PA, is only marginally involved in clot-lysis because its presence and activity is mostly confined to tissues. Here, u-PA and plasmin are involved in the proteolysis of matrix proteins during processes like tissue repair and cell migration. The activities of both t-PA and u-PA are controlled by their specific inhibitor, plasminogen activator inhibitor-1 (PAI-1), the main topic of this review. PAI-1 was first purified as a protein secreted by endothelial

Regulation of arterial thrombolysis by plasminogen activator inhibitor-1 in mice.

Platelet-rich arterial thrombi are resistant to lysis by plasminogen activators. However, the mechanisms underlying thrombolysis resistance are poorly defined. Plasminogen activator inhibitor-1 (PAI-1), which is present in plasma, platelets, and vascular endothelium, may be an important determinant of the resistance of arterial thrombi to lysis. However, in vitro studies examining the regulation of platelet-rich clot lysis by PAI-1 have yielded inconsistent results. We developed a murine arterial injury model and applied it to wild-type (PAI-1 [+/+]) and PAI-1-deficient (PAI-1 [-/-]) animals. FeCl3 was used to induce carotid artery thrombosis. Thrombi consisted predominantly of dense platelet aggregates, consistent with the histology of thrombi in large-animal arterial injury models and human acute coronary syndromes. To examine the role of PAI-1 in regulating endogenous clearance of platelet-rich arterial thrombi, thrombi were induced in 22 PAI-1 (+/+) mice 14 PAI-1 (-/-) mice. Twenty-four hours later, the amount of residual thrombus was determined by histological analysis of multiple transverse sections of each artery. Residual thrombus was detected in 55 of 85 sections (64.7%) obtained from PAI-1 (+/+) mice compared with 19 of 56 sections (33.9%) from PAI-1 (-/-) mice (P=.009). Computer-assisted planimetry analysis revealed that mean thrombus cross-sectional area was 0.033+/-0.0271 mm2 in PAI-1 (+/+) mice versus 0.016+/-0.015 mm2 in PAI-1 (-/-) mice (P=.048). PAI-1 is an important determinant of thrombolysis at sites of arterial injury. Application of this model to other genetically altered mice should prove useful for studying the molecular determinants of arterial thrombosis and thrombolysis.

RGDFAP

As published in a recent issue of Blood Coagulation and Fibrinolysis, the hybrid peptide RGDFAP, composed of RGDF (Arg-Gly-Asp-Phe) coupled to a synthetic peptide residue of the carboxy terminal part of antiplasmin (AP26) inhibited platelet activation and augmented plasmin generation and in vitro fibrin clot lysis. This peptide contains an RGD motif which provides linkage to platelet GP IIb-IIIa. The antiplasmin part of the molecule may attach free plasminogen, which in turn increases the amount of platelet surface bound plasminogen, probably yielding enhanced lytic action at the site of thrombus formation. This hypothesis was investigated and confirmed by the results of platelet-plasminogen binding assays, using FITC-labelled antiplasmin antibodies and radioligand binding analysis. Increased platelet-linked plasminogen was detected by a chromogenic method, along with the acceleration of in vitro lysis of platelet-rich clots in the presence of RGDFAP peptide.

Peptide-mediated inactivation of recombinant and platelet plasminogen activator inhibitor-1 in vitro.

Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA) and urokinase plasminogen activator, is an important regulator of the blood fibrinolytic system. Elevated plasma levels of PAI-1 are associated with thrombosis, and high levels of PAI-1 within platelet-rich clots contribute to their resistance to lysis by t-PA. Consequently, strategies aimed at inhibition of PAI-1 may prove clinically useful. This study was designed to test the hypothesis that a 14-amino acid peptide, corresponding to the PAI-1 reactive center loop (residues 333-346), can rapidly inhibit PAI-1 function. PAI-1 (0.7 microM) was incubated with peptide (55 microM) at 37 degrees C. At timed intervals, residual PAI-1 activity was determined by addition of reaction mixture samples to t-PA and chromogenic substrate. The T1/2 of PAI-1 activity in the presence of peptide was 4 +/- 3 min compared to a control T1/2 of 98 +/- 18 min. The peptide also inhibited complex formation between PAI-1 and t-PA as demonstrated by SDS-PAGE analysis. However, the capacity of the peptide to inhibit PAI-1 bound to vitronectin, a plasma protein that stabilizes PAI-1 activity, was markedly attenuated. Finally, the peptide significantly enhanced in vitro lysis of platelet-rich clots and platelet-poor clots containing recombinant PAI-1. These results indicate that a 14-amino acid peptide can rapidly inactivate PAI-1 and accelerate fibrinolysis in vitro. These studies also demonstrate that PAI-1 function can be directly attenuated in a physiologic setting and suggest a novel approach for augmenting fibrinolysis in vivo.

The role of PAI-1 in the lysis of platelet rich clots

The role of PAI-1 in the lysis of platelet rich clots