Michael H. Kroll

Nitric Oxide Induces Heme Oxygenase-1 Gene Expression and Carbon Monoxide Production in Vascular Smooth Muscle Cells

Since recent studies demonstrate that vascular smooth muscle cells synthesize two distinct guanylate cyclase-stimulatory gases, NO and CO, we examined possible regulatory interactions between these two signaling molecules. Treatment of rat aortic smooth muscle cells with the NO donors, sodium nitroprusside, S-nitroso-N-acetyl-penicillamine, or 3-morpholinosydnonimine, increased heme oxygenase-I (HO-1) mRNA and protein levels in a concentration and time-dependent manner. Both actinomycin D and cycloheximide blocked NO-stimulated HO-1 mRNA and protein expression. Nuclear run-on experiments demonstrated that NO donors increased HO-1 gene transcription between 3- and 6-fold. In contrast, NO donors had no effect on the stability of HO-1 mRNA. Incubation of vascular smooth muscle cells with the membrane-permeable cGMP analogues, dibutyryl cGMP and 8-bromo-cGMP, failed to induce HO-1 gene expression. Treatment of vascular smooth muscle cells with NO donors also stimulated the production and release of CO, as demonstrated by the CO-dependent increase in intracellular cGMP levels in coincubated platelets. Finally, incubating vascular smooth muscle cells with interleukin-1 beta and tumor necrosis factor-alpha induced NO synthesis and also significantly increased the level of HO-1 protein. The cytokine-stimulated production of both NO and HO-1 protein in smooth muscle cells was blocked by the NO synthase inhibitor methyl-L-arginine. These results demonstrate that exogenously administered or endogenously released NO stimulates HO-1 gene expression and CO production in vascular smooth muscle cells. The ability of NO to induce HO-catalyzed CO release from vascular smooth muscle cells provides a novel mechanism by which NO might modulate soluble guanylate cyclase and, thereby, vascular smooth muscle cell and platelet function.

von Willebrand factor binding to platelet GpIb initiates signals for platelet activation.

The hypothesis that von Willebrand factor (vWF) binding to platelet membrane glycoprotein Ib (GpIb) initiates intracellular pathways of platelet activation was studied. We measured the biochemical responses of intact human platelets treated with ristocetin plus vWF multimers purified from human cryoprecipitate. vWF plus ristocetin causes the breakdown of phosphatidylinositol 4,5-bisphosphate, the production of phosphatidic acid (PA), the activation of protein kinase C (PKC), increase of ionized cytoplasmic calcium ([Ca2+]i), and the synthesis of thromboxane A2. PA production, PKC activation, and the rise of [Ca2+]i stimulated by the ristocetin-induced binding of vWF multimers to platelets are inhibited by an anti-GpIb monoclonal antibody, but are unaffected by anti-GpIIb-IIIa monoclonal antibodies. Indomethacin also inhibits these responses without impairing platelet aggregation induced by vWF plus ristocetin. These results indicate that vWF binding to platelets initiates specific intraplatelet signaling pathways. The mechanism by which this occurs involves an arachidonic acid metabolite-dependent activation of phospholipase C after vWF binding to platelet membrane GpIb. This signal then causes PKC activation and increases of [Ca2+]i, which promote platelet secretion and potentiate aggregation.

Vascular Smooth Muscle Cell Heme Oxygenases Generate Guanylyl Cyclase–Stimulatory Carbon Monoxide

BACKGROUND Carbon monoxide (CO), like nitric oxide (NO), stimulates soluble guanylyl cyclase and thereby raises intracellular levels of cGMP. We examined the endogenous capacity of vascular smooth muscle cells (SMCs) to produce CO from heme through the activity of heme oxygenases. METHODS AND RESULTS Cultured SMCs from rat aorta (RASMCs) expressed immunoreactive inducible heme oxygenase-1 (HO-1) and constitutive HO-2. Treatment of RASMCs with hemin and sodium arsenite, which are inducers of HO-1, stimulated RASMC cGMP without stimulating nitrite release or inducible NO synthase expression, and the induced elevations of cGMP were not inhibited by the NO synthase inhibitor NG-methyl-L-arginine. Induced CO from RASMCs likewise caused elevation of cGMP levels in platelets coincubated with the vascular cells. Zinc protoporphyrin IX, an inhibitor of HO, reversed the inducible increases in platelet cGMP. CONCLUSIONS These results indicate that vascular SMCs have both constitutive and inducible HO activity, and they respond to specific stimuli to generate guanylyl cyclase-stimulatory CO in the same SMCs and in coincubated platelets.

Platelets increase the proliferation of ovarian cancer cells

Platelets promote metastasis and angiogenesis, but their effect on tumor cell growth is uncertain. Here we report a direct proliferative effect of platelets on cancer cells both in vitro and in vivo. Incubation of platelets with ovarian cancer cells from murine (ID8 and 2C6) or human (SKOV3 and OVCAR5) origin increased cell proliferation. The proliferative effect of platelets was not dependent on direct contact with cancer cells, and preincubation of platelets with blocking antibodies against platelet adhesion molecules did not alter their effect on cancer cells. The proliferative effect of platelets was reduced by fixing platelets with paraformaldehyde, preincubating platelets with a TGF-β1-blocking antibody, or reducing expression of TGF-βR1 receptor on cancer cells with siRNA. Infusing platelets into mice with orthotopic ovarian tumors significantly increased the proliferation indices in these tumors. Ovarian cancer patients with thrombocytosis had higher tumor proliferation indices compared with patients with normal platelet counts.

Partial ADAMTS13 deficiency in atypical hemolytic uremic syndrome

Complement dysregulation leads to atypical hemolytic uremic syndrome (aHUS), while ADAMTS13 deficiency causes thrombotic thrombocytopenic purpura. We investigated whether genetic variations in the ADAMTS13 gene partially explain the reduced activity known to occur in some patients with aHUS. We measured complement activity and ADAMTS13 function, and completed mutation screening of multiple complement genes and ADAMTS13 in a large cohort of aHUS patients. In over 50% of patients we identified complement gene mutations. Surprisingly, 80% of patients also carried at least 1 nonsynonymous change in ADAMTS13, and in 38% of patients, multiple ADAMTS13 variations were found. Six of the 9 amino acid substitutions in ADAMTS13 were common single nucleotide polymorphisms; however, 3 variants-A747V, V832M, and R1096H- were rare, with minor allele frequencies of 0.0094%, 0.5%, and 0.32%, respectively. Reduced complement and ADAMTS13 activity (<60% of normal activity) were found in over 60% and 50% of patients, respectively. We concluded that partial ADAMTS13 deficiency is a common finding in aHUS patients and that genetic screening and functional tests of ADAMTS13 should be considered in these patients.

Direct demonstration of radiolabeled von willebrand factor binding to platelet glycoprotein Ib and IIb-IIIa in the presence of shear stress

In this study it is demonstrated for the first time that shear stress induces the binding of exogenous von Willebrand factor (vWF) multimers to platelets. The vWF preparations used were:125I-vWF purified from human cryoprecipitate (and including all vWF multimers present in normal plasma); and35S-cysteine-vWF secreted by human umbilical vein endothelial cells (HUVECs) (and containing unusually large vWF forms, as well as all plasma-type vWF multimers). Direct shear-induced binding to washed platelets (300–360×103/μl) of radiolabeled vWF was maximum at 60–120 dynes/cm2 evaluated at 30 sec and was in extent about one-quarter of the binding stimulated by ristocetin after 3 min of incubation. The shear-induced binding of only a small percentage of added radiolabeled vWF was sufficient to initiate aggregation. Radiolabeled vWF attached to both glycoprotein (GP) Ib and GPIIb-IIIa receptors in the shear field, with complete inhibition of binding occurring with simultaneous blockade of both receptors. Binding was potentiated by ADP released from sheared platelets.

Von Willebrand Factor is a cofactor in complement regulation

Several complement proteins interact with hemostatic factors. We discovered that von Willebrand factor (VWF) acts as a cofactor for factor I-mediated cleavage of complement C3b, thereby shutting down complement activation. The complement regulatory function of VWF multimers depends on their size. Smaller VWF multimers enhance cleavage of C3b but large and ultra-large VWF (ULVWF) multimers have no effect on C3b cleavage and permit default complement activation. We conclude that normal plasma VWF multimers prevent complement activation and steer the complement pathway toward generation of inactivated C3b (iC3b). ULVWF multimers, as are present in patients with thrombotic microangiopathy, lack an inhibitory effect on complement and permit complement activation.

Fibrinolysis Inhibits Shear Stress–Induced Platelet Aggregation

BACKGROUND Shear stress-induced platelet aggregation may initiate arterial thrombosis at sites of pathological blood flow. Shear stress-induced platelet aggregation is mediated by von Willebrand factor (vWf) binding to platelet membrane glycoprotein (GP) Ib and GP IIb/IIIa. Tissue-type plasminogen activator (TPA) induces thrombolysis in coronary arteries through the local generation of plasmin. Plasmin also proteolyses GP Ib and plasma vWf. METHODS AND RESULTS Because these effects could mitigate shear stress-induced platelet aggregation, we investigated the effect of fibrinolytic agents on platelet aggregation in response to a pathological shear stress of 120 dynes/cm2 generated by a cone-and-platen rotational viscometer. Plasmin inhibited shear stress-induced aggregation of washed platelets, and this was associated with a decrease in GP Ib. TPA, at concentrations > or = 2000 IU/mL, significantly inhibited shear stress-induced platelet aggregation of platelet-rich plasma without a decrease in platelet GP Ib. In plasma-platelet mixing experiments, we determined that the TPA effect was localized to plasma. Purified vWf multimer degradation by TPA (in the presence of exogenous plasminogen) was associated with the loss of the capacity of vWf to support shear stress-induced platelet aggregation. CONCLUSIONS These results demonstrate that TPA inhibits platelet aggregation in response to pathological shear stress by altering the multimeric composition of vWf. This effect of TPA on shear stress-induced platelet aggregation may contribute, along with fibrinolysis, to the therapeutic effect of TPA in restoring blood flow during acute coronary artery thrombosis.

Venous thromboembolism (VTE) rates following the implementation of extended duration prophylaxis for patients undergoing surgery for gynecologic malignancies.

OBJECTIVE To compare the incidence of venous thromboembolism (VTE) before and after the implementation of standardized extended duration prophylaxis guidelines in women undergoing laparotomy for gynecologic cancer. METHODS In October 2009, departmental practice guidelines were implemented for VTE prevention. Patients undergoing laparotomy for gynecologic cancer were started on low molecular weight heparin (LMWH) within 24h of surgery and it was continued for a total of 28 days postoperatively. The incidence of VTE diagnosed within 30 and 90 days of surgery was determined and compared to a historic cohort of patients who underwent surgery prior to implementation of the guidelines. RESULTS The incidence of VTE within 30 days of surgery decreased from 2.7% (8/300) to 0.6% (2/334) following implementation of VTE prevention guidelines (78% reduction, p=0.040). However, when the pre and post-guideline implementation groups were compared for the development of VTE within 90 days of surgery, there was no significant difference (11/300 (3.7%) vs. 10/334 (3.0%) respectively, p=0.619). The median time between surgery and VTE diagnosis was 12 days in the pre-guideline implementation group, compared with 57 days in the post-guideline implementation group (p=0.012). CONCLUSION Patients receiving extended duration LMWH were found to have significantly lower rates of VTE within 30 days of surgery when compared with similar patients who did not receive extended duration LMWH. However, this effect was not sustained when the groups were compared for VTE diagnosis within 90 days of surgery. Additional study is needed to further reduce long-term VTE rates in this high-risk population.

AIDS-Related Systemic Non-Hodgkin's Lymphoma at a Large Community Program

The introduction of triple antiretroviral therapy has led to reductions in opportunistic diseases in HIV-infected patients. However, little is known of the effect of this therapy on the clinical and pathological features and the outcome of patients with AIDS-related systemic non-Hodgkins lymphoma (NHL). We examined the incidence and clinical manifestations of HIV-infected patients with systemic NHL at the Harris County Hospital District and Veterans Affairs Medical Center (Houston, TX). Between January 1996 and December 2000, 76 cases of systemic NHL were diagnosed in 3655 HIV-infected patients. Three groups of patients diagnosed with systemic NHL were identified according to their history of antiretroviral therapy: treatment naive (n = 20), dual nucleoside (n = 22), and triple antiretroviral drug-treated patients (n = 34). The median duration of antiretroviral therapy before the diagnosis of systemic NHL in the triple antiretroviral and dual nucleoside treatment groups was 12 versus 8 months (p < 0.0004). Thirty-five percent of patients (12 of 34) in the triple treatment group had an HIV RNA viral load of <400 copies/ml and their median CD4+ cell count was 301 cells/mm(3) (range, 46 to 667 cells/mm(3)) at the time of diagnosis of systemic NHL. More patients treated with triple antiretroviral therapy received complete courses of chemotherapy as compared with the other two groups (p = 0.013). However, the overall survival did not differ significantly among the three groups of patients. These data suggest that AIDS-related systemic NHL continues to occur even in patients treated with triple antiretroviral therapy. In addition, this opportunistic malignancy is associated with significant mortality. Therefore, it is necessary to develop a better understanding of the pathogenesis of this disease.