Dataset Information

Identification and Characterization of Plasmin-Independent Thrombolytic Enzymes.

ABSTRACT:

Rationale

Current thrombolytic agents activate plasminogen to plasmin which triggers fibrinolysis to dissolve thrombi. Since plasmin is a nonspecific proteolytic enzyme, all of the current plasmin-dependent thrombolytics lead to serious hemorrhagic complications, demanding a new class of fibrinolytic enzymes independent from plasmin activation and undesirable side effects. We speculated that the mammalian version of bacterial heat-shock proteins could selectively degrade intravascular thrombi, a typical example of a highly aggregated protein mixture.

Objective

The objective of this study is to identify enzymes that can dissolve intravascular thrombi specifically without affecting fibrinogen and fibronectin so that the wound healing processes remain uninterrupted and tissues are not damaged. In this study, HtrA (high-temperature requirement A) proteins were tested for its specific proteolytic activity on intravascular thrombi independently from plasmin activation.

Methods and results

HtrA1 and HtrA2/Omi proteins, collectively called as HtrAs, lysed ex vivo blood thrombi by degrading fibrin polymers. The thrombolysis by HtrAs was plasmin-independent and specific to vascular thrombi without causing the systemic activation of plasminogen and preventing nonspecific proteolysis of other proteins including fibrinogen and fibronectin. As expected, HtrAs did not disturb clotting and wound healing of excised wounds from mouse skin. It was further confirmed in a tail bleeding and a rebleeding assay that HtrAs allowed normal clotting and maintenance of clot stability in wounds, unlike other thrombolytics. Most importantly, HtrAs completely dissolved blood thrombi in tail thrombosis mice, and the intravenous injection of HtrAs to mice with pulmonary embolism completely dissolved intravascular thrombi and thus rescued thromboembolism.

Conclusions

Here, we identified HtrA1 and HtrA2/Omi as plasmin-independent and highly specific thrombolytics that can dissolve intravascular thrombi specifically without bleeding risk. This work is the first report of a plasmin-independent thrombolytic pathway, providing HtrA1 and HtrA2/Omi as ideal therapeutic candidates for various thrombotic diseases without hemorrhagic complications.

SUBMITTER: Hassan MM

PROVIDER: S-EPMC7861503 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Publications

Identification and Characterization of Plasmin-Independent Thrombolytic Enzymes.

Hassan Md Mehedi MM Sharmin Shirina S Kim Hyeon-Jin HJ Hong Seong-Tshool ST

Circulation research 20201209 3

<h4>Rationale</h4>Current thrombolytic agents activate plasminogen to plasmin which triggers fibrinolysis to dissolve thrombi. Since plasmin is a nonspecific proteolytic enzyme, all of the current plasmin-dependent thrombolytics lead to serious hemorrhagic complications, demanding a new class of fibrinolytic enzymes independent from plasmin activation and undesirable side effects. We speculated that the mammalian version of bacterial heat-shock proteins could selectively degrade intravascular th ...[more]

PMID: 33292062

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Project description:BACKGROUND:Hyperfibrinolysis plays an integral role in the genesis of trauma-induced coagulopathy. Recent data demonstrate that red blood cell lysis promotes fibrinolysis; however, the mechanism is unclear. Hemoglobin-based oxygen carriers (HBOCs) have been developed for resuscitation and have been associated with coagulopathy. We hypothesize that replacement of whole blood (WB) using an HBOC results in a coagulopathy because of the presence of free hemoglobin. MATERIALS AND METHODS:WB was sampled from healthy donors (n = 6). The clotting profile of each citrated sample was evaluated using native thromboelastography. Serial titrations were performed using both HBOC (PolyHeme) and normal saline (NS; 5%, 25%, and 50%) and evaluated both with and without a 75-ng/mL tissue plasminogen activator (tPA) challenge. Tranexamic acid (TXA) was added to inhibit plasmin-dependent fibrinolysis. Fibrinolysis was measured and recorded as lysis at 30 min (LY30), the percentage of clot LY30 after maximal clot strength. Dilution of WB with NS or HBOC was correlated using LY30 via Spearman rho coefficients. Groups were also compared using a Friedman test and post hoc analysis with a Bonferroni adjustment. RESULTS:tPA-provoked fibrinolysis was enhanced by both HBOC (median LY30 at 5%, 25%, and 50% titrations: 11%, 21%, and 44%, respectively; Spearman = 0.94; P < 0.001) and NS (11%, 28%, and 58%, respectively; Spearman = 0.790; P < 0.001). However, HBOC also enhanced fibrinolysis without the addition of tPA (1%, 4%, 5%; Spearman = 0.735; P = 0.001) and NS did not (1%, 2%, 1%; r = 0.300; P = 0.186. Moreover, addition of TXA did not alter or inhibit this fibrinolysis (WB versus 50% HBOC: 1.8% versus 5.7%, P = 0.04). There was no significant difference in fibrinolysis of HBOC with or without TXA (50% HBOC versus 50% HBOC + TXA: 5.6% versus 5.7%, P = 0.92). In addition, the increased fibrinolysis seen with NS was reversed when TXA was present (WB versus 50% NS: 1.8% versus 1.7%, P = 1.0). CONCLUSIONS:HBOCs enhance fibrinolysis both with and without addition of tPA; moreover, this mechanism is independent of plasmin as the phenomenon persists in the presence of TXA. Our findings indicate the hemoglobin molecule or its components stimulate fibrinolysis by both tPA-dependent and innate mechanisms.

Dataset Information

Identification and Characterization of Plasmin-Independent Thrombolytic Enzymes.

Rationale

Objective

Methods and results

Conclusions

Publications

Identification and Characterization of Plasmin-Independent Thrombolytic Enzymes.

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