Yihua Zhou

Murine prolylcarboxypeptidase depletion induces vascular dysfunction with hypertension and faster arterial thrombosis

Prolylcarboxypeptidase (PRCP) activates prekallikrein to plasma kallikrein, leading to bradykinin liberation, and degrades angiotensin II. We now identify PRCP as a regulator of blood vessel homeostasis. β-Galactosidase staining in PRCP(gt/gt) mice reveals expression in kidney and vasculature. Invasive telemetric monitorings show that PRCP(gt/gt) mice have significantly elevated blood pressure. PRCP(gt/gt) mice demonstrate shorter carotid artery occlusion times in 2 models, and their plasmas have increased thrombin generation times. Pharmacologic inhibition of PRCP with Z-Pro-Prolinal or plasma kallikrein with soybean trypsin inhibitor, Pro-Phe-Arg-chloromethylketone or PKSI 527 also shortens carotid artery occlusion times. Aortic and renal tissues have uncoupled eNOS and increased reactive oxygen species (ROS) in PRCP(gt/gt) mice as detected by dihydroethidium or Amplex Red fluorescence or lucigenin luminescence. The importance of ROS is evidenced by the fact that treatment of PRCP(gt/gt) mice with antioxidants (mitoTEMPO, apocynin, Tempol) abrogates the hypertensive, prothrombotic phenotype. Mechanistically, our studies reveal that PRCP(gt/gt) aortas express reduced levels of Kruppel-like factors 2 and 4, thrombomodulin, and eNOS mRNA, suggesting endothelial cell dysfunction. Further, PRCP siRNA treatment of endothelial cells shows increased ROS and uncoupled eNOS and decreased protein C activation because of thrombomodulin inactivation. Collectively, our studies identify PRCP as a novel regulator of vascular ROS and homeostasis.

Oral thrombostatin FM19 inhibits prostate cancer.

Thrombin stimulates proliferation, invasion and metastasis by cleaving protease-activated receptor 1 (PAR1) on human prostate cancer cells. Current direct thrombin inhibitors pose risks for bleeding in the cancer patients. We have developed an oral reversible direct thrombin inhibitor called FM19. FM19 inhibits thrombin-induced calcium mobilisation of PC3 cells with an IC50 of 15 μM with a 95% confidence interval of 7.3-31.6 μM. Thrombin stimulation increases PC3 cell invasion three-fold from 27.1 ± 11.4 to 66 ± 11.6. FM19 or bivalirudin reduces cell invasion at ≥0.1 μM (p≤0.02). After inoculation with PC3 cells, nude mice were treated with oral FM19 at 3 mg/ml in the drinking water. The treated mice did not have long bleeding times and only a 1.4-fold increase in their thrombin clotting time. However, with treatment, the mice have a reduced rate of tumour growth 0.26 ± 0.17 fold change/day vs. 0.55 ± 0.35 for untreated (p = 0.038), reduced fold change in tumour size 5.3 ± 0.47 to 8.9 ± 1.8 (untreated) (p=0.048), and reduced overall tumour weight 0.5 ± 0.31 g vs. 0.82 ± 0.32 g (untreated) (p=0.04). On microscopic examination, FM19 treatment reduces the number of large vessels in the tumours from 4.6 ± 2.1 per high-powered field in untreated samples to 1.4 ± 1.4 in treated samples (p≤0.04). These studies show FM19 reduces prostate tumour growth in vivo at a concentration below that needed for anticoagulation. These data suggest novel opportunities for oral direct thrombin inhibitors in cancer therapy.

Thrombostatin FM compounds: direct thrombin inhibitors - mechanism of action in vitro and in vivo.

Summary.  Background: Novel pentapeptides called Thrombostatin FM compounds consisting mostly of D‐isomers and unusual amino acids were prepared based upon the stable angiotensin converting enzyme breakdown product of bradykinin – RPPGF. Methods and Results: These peptides are direct thrombin inhibitors prolonging the thrombin clotting time, activated partial thromboplastin time, and prothrombin time at ≥0.78, 1.6, and 1.6 μm, respectively. They competitively inhibit α‐thrombin‐induced cleavage of a chromogenic substrate at 4.4–8.2 μm. They do not significantly inhibit plasma kallikrein, factor (F) XIIa, FXIa, FIXa, FVIIa‐TF, FXa, plasmin or cathepsin G. One form, FM19 [rOicPaF(p‐Me)], blocks α‐thrombin‐induced calcium flux in fibroblasts with an IC50 of 6.9 ± 1.2 μm. FM19 achieved 100% inhibition of threshold α‐ or γ‐thrombin‐induced platelet aggregation at 8.4 ± 4.7 μm and 16 ± 4 μm, respectively. The crystal structure of thrombin in complex with FM19 shows that the N‐terminal D‐Arg retrobinds into the S1 pocket, its second residue Oic interacts with His‐57, Tyr‐60a and Trp‐60d, and its C‐terminal p‐methyl Phe engages thrombin’s aryl binding site composed of Ile‐174, Trp‐215, and Leu‐99. When administered intraperitoneal, intraduodenal, or orally to mice, FM19 prolongs thrombin clotting times and delays carotid artery thrombosis. Conclusion: FM19, a low affinity reversible direct thrombin inhibitor, might be useful as an add‐on agent to address an unmet need in platelet inhibition in acute coronary syndromes in diabetics and others who with all current antiplatelet therapy still have reactive platelets.