Search

Hematopoietic stem cell transplantation following myeloablative chemotherapy is a curative treatment for many hematopoietic malignancies. However, profound granulocytopenia during the interval between transplantation and marrow recovery exposes recipients to risks of fatal infection, a significant source of transplant-associated morbidity and mortality. We have previously described the discovery of a small molecule, SW033291, that potently inhibits the prostaglandin degrading enzyme 15-PGDH, increases bone marrow prostaglandin E2, and accelerates hematopoietic recovery following murine transplant. Here we describe the efficacy of (+)-SW209415, a second-generation 15-PGDH inhibitor, in an expanded range of models relevant to human transplantation. (+)-SW209415 is 10,000-fold more soluble, providing the potential for intravenous delivery, while maintaining potency in inhibiting 15-PGDH, increasing in vivo prostaglandin E2, and accelerating hematopoietic regeneration following transplantation. In additional models, (+)-SW209415: (i) demonstrated synergy with granulocyte colony-stimulating factor, the current standard of care; (ii) maintained efficacy as transplant cell dose was escalated; (iii) maintained efficacy when transplant donors and recipients were aged; and (iv) potentiated homing in xenotransplants using human hematopoietic stem cells. (+)-SW209415 showed no adverse effects, no potentiation of in vivo growth of human myeloma and leukemia xenografts, and, on chronic high-dose administration, no toxicity as assessed by weight, blood counts and serum chemistry. These studies provide independent chemical confirmation of the activity of 15-PGDH inhibitors in potentiating hematopoietic recovery, extend the range of models in which inhibiting 15-PGDH demonstrates activity, allay concerns regarding potential for adverse effects from increasing prostaglandin E2, and thereby, advance 15-PGDH as a therapeutic target for potentiating hematopoietic stem cell transplantation.

How single-chain urokinase (ScuPA) mediates angiogenesis is incompletely understood. ScuPA (≥4 nM) induces phosphorylated (p)ERK1/2 (MAPK44 and MAPK42) and pAkt (Ser473) in umbilical vein and dermal microvascular endothelial cells. Activation of pERK1/2 by ScuPA is blocked by PD-98059 or U-0126, and pAkt (Ser473) activation is inhibited by wortmannin or LY-294002. ScuPA (32 nM) or protease-inhibited two-chain urokinase stimulates pERK1/2 to the same extent, indicating that signaling is not dependent on enzymatic activity. ScuPA induces pERK1/2, but not pAkt (Ser473), in SIN1−/−cells, indicating that the two pathways are not identical. Peptides from domain 2 of the urokinase plasminogen activator receptor (uPAR) or domain 5 of high-molecular-weight kininogen compete with ScuPA for the induction of pERK1/2 and pAkt (Ser473). A peptide of the integrin-binding site on uPAR, a β1-integrin peptide that binds uPAR, antibody 6S6 to β1-integrin, tyrosine kinase inhibitors AG-1478 or PP3, and small interfering RNA knockdown of VEFG receptor 2, but not HER1–HER4, blocked ScuPA-induced pERK1/2 and pAkt (Ser473). ScuPA-induced endothelial cell proliferation was blocked by inhibitors of pERK1/2 and pAkt (Ser473), antibody 6S6, and uPAR or kininogen peptides. ScuPA initiated aortic sprouts and Matrigel plug angiogenesis in normal, but not uPAR-deficient, mouse aortae or mice, respectively, but these were blocked by PD-98059, LY-294002, AG-1478, or cleaved high-molecular-weight kininogen. In summary, this investigation indicates a novel, a nonproteolytic signaling pathway initiated by zymogen ScuPA and mediated by domain 2 of uPAR, β1-integrins, and VEGF receptor 2 leading to angiogenesis. Kininogens or peptides from it downregulate this pathway.

Bradykinin B2 receptor-deleted mice (Bdkrb2(-/-)) have delayed carotid artery thrombosis times and prolonged tail bleeding time resulting from elevated angiotensin II (AngII) and angiotensin receptor 2 (AT2R) producing increased plasma nitric oxide (NO) and prostacyclin. Bdkrb2(-/-) also have elevated plasma angiotensin-(1-7) and messenger RNA and protein for its receptor Mas. Blockade of Mas with its antagonist A-779 in Bdkrb2(-/-) shortens thrombosis times (58 ± 4 minutes to 38 ± 4 minutes) and bleeding times (170 ± 13 seconds to 88 ± 8 seconds) and lowers plasma nitrate (22 ± 4 μM to 15 ± 5 μM), and 6-keto-PGF1α (259 ± 103 pg/mL to 132 ± 58 pg/mL). Bdkrb2(-/-) platelets express increased NO, guanosine 3',5'-cyclic monophosphate, and cyclic adenosine monophosphate with reduced spreading on collagen, collagen peptide GFOGER, or fibrinogen. In vivo A-779 or combined L-NAME and nimesulide treatment corrects it. Bdkrb2(-/-) platelets have reduced collagen-related peptide-induced integrin α2bβ3 activation and P-selectin expression that are partially corrected by in vivo A-779, nimesulide, or L-NAME. Bone marrow transplantations show that the platelet phenotype and thrombosis time depends on the host rather than donor bone marrow progenitors. Transplantation of wild-type bone marrow into Bdkrb2(-/-) hosts produces platelets with a spreading defect and delayed thrombosis times. In Bdkrb2(-/-), combined AT2R and Mas overexpression produce elevated plasma prostacyclin and NO leading to acquired platelet function defects and thrombosis delay.

SummaryThrombin 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 threefold 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.

SummaryRatnoff and his coworkers recognised that factor XII (XII) stimulates cell growth and activates mitogen-activated protein kinase. We determined the receptor(s) for this function and the consequence of this signalling pathway. Investigations show that the urokinase plasminogen activator receptor serves as the XII binding site on cultured umbilical vein endothelial cells. When XII binds, it stimulates ERK1/2 and Akt S473 phosphorylation. These events are distinct because when cell mTORC2 is absent, XII phosphorylates ERK1/2 but not Akt S473. Zymogen XII is an equal stimulator of signalling as XIIa or inhibitor-treated XIIa. Peptides from uPAR domain 2 block XII binding and ERK1/2 and Akt phosphorylation. Furthermore, antibodies to the integrins β1 and α5 block XII signalling. Likewise, inhibitors to the EGFR block XII-induced phosphorylation events. XII stimulates cell growth and proliferation. XII induces angiogenesis ex vivo in normal aortic sprouts and in vivo in matrigel plugs in normal mice, but not in aorta from uPAR knockout mice or matrigel plugs placed into uPAR-deleted mice. Skin biopsies constitutively or in a wound nine days after injury show reduced CD31 antigen expression in specimens from XII knockout mice compared to wild-type mice. These studies indicate that XII stimulates angiogenesis, a physiologic function independent of contact activation.

Hdm2 is elevated in numerous types of malignancies and is thought to impede the function of wild-type p53. Reactivation of p53 by disrupting the association with Hdm2 was the impetus for the development of Nutlin3. Although regulation of p53 has been the central focus of Hdm2 activity, it also binds other proteins through its p53-binding domain. Here, we show that hypoxia-inducible factor 1α (HIF1α) binds to Hdm2 in the domain designated to bind p53. HIF1α and p53 share a conserved motif that is required to bind Hdm2. Distinct complexes form between Hdm2-HIF1α and Hdm2-p53 as determined by immunoprecipitation of nuclear extracts and in vitro. The Hdm2 antagonist Nutlin3 prevents the association between Hdm2 and HIF1α. The vascular endothelial growth factor (VEGF) gene is a transcriptional target of HIF1α, and under normoxic or hypoxic conditions, Hdm2 increases HIF1α activity to induce VEGF production. Blocking the association of Hdm2 and HIF1α by Nutlin3, or ablating Hdm2 expression, diminished the level of VEGF under conditions of normoxia or hypoxia. Our findings establish a unique role for Nutlin3 in attenuating VEGF induction by preventing the association of Hdm2 with HIF1α. [Cancer Res 2007;67(2):450–4]

A shot in the arm for damaged tissue Tissue damage can be caused by injury, disease, and even certain medical treatments. There is great interest in identifying drugs that accelerate tissue regeneration and recovery, especially drugs that might benefit multiple organ systems. Zhang et al. describe a compound with this desired activity, at least in mice (see the Perspective by FitzGerald). SW033291 promotes recovery of the hematopoietic system after bone marrow transplantation, prevents the development of ulcerative colitis in the intestine, and accelerates liver regeneration after hepatic surgery. It acts by inhibiting an enzyme that degrades prostaglandins, lipid signaling molecules that have been implicated in tissue stem cell maintenance. Science , this issue 10.1126/science.aaa2340 ; see also p. 1208

Hageman trait refers to an inherited deficiency of coagulation factor XII (FXII). The term was named by Oscar Ratnoff in 1955 after the index patient John Hageman, who had ‘incoagulable’ blood in vitro but no abnormal bleeding even after surgeries [1, 2]. Subsequently, Earl Davie and Oscar Ratnoff isolated Hageman factor, the protein missing in Hageman’s blood, which later was called FXII [3, 4]. This pioneer work contributed to the ‘Waterfall/Cascade’ hypothesis of blood coagulation presented in 1964 [5, 6], which established the basic principle that blood coagulation is mediated by the sequential activation of a series of plasma proteases. John Hageman died of pulmonary thromboembolism during bed rest from a broken hip in 1968 before gene cloning techniques were available [7]. As a result, the genetic defect underlying his FXII-deficiency was never determined. In this study, we obtained blood samples from descendants of the Hageman family to analyze their F12 gene that encodes FXII. The study was approved by the Cleveland Clinic Institutional Review Board. All participants provided written consent. John Hageman was never married and had no known descendants (Fig. 1A). We obtained blood samples from III-1, III-3-5 and IV-3-5, who were family members of his siblings’ descendants. None of these individuals had a history of abnormal bleeding. PCR and DNA sequencing of all 14 F12 gene exons in these individuals found no deletion, insertion or non-synonymous point mutations. In III-3 and IV-5, however, a G→A intronic mutation was identified at nucleotide position 11396 of the F12 gene [8] (Fig. 1B), which was part of the splice acceptor site of exon 14 (Fig. 1C). The mutation abolished the original splice acceptor site and created a new site one nucleotide downstream. As a result, the exon 14 reading frame was shifted by one position, thereby encoding a new peptide that replaced the catalytic serine and the rest of the protease domain (Fig. 1C). Fig. 1 (A) Hageman family pedigree. The pedigree was modified for confidentiality. J.H., John Hageman. Diamonds with a slash line represent deceased individuals. Diamonds with a dot represent heterozygous carriers. IV-1–2 were not available for the study. ... Interestingly, this 11396(G→A) mutation was reported previously in FXII-deficient individuals in Germany [9, 10]. PCR analysis of transcripts from the mutant F12 gene confirmed the predicted fusion mRNA. The study also showed that the fusion protein, if translated from the fusion mRNA, was unstable, because individuals homozygous for this mutation had no detectable plasma FXII antigen or activity [9, 10]. By ELISA (Innovative Research Inc.), we found that plasma FXII antigen levels in III-3 and IV-5, who were heterozygous for the mutation, were ~50% of that in other members of the family (Fig. 1A). The antigen level in the normal individuals of this family was ~1.8 fold higher than that of pooled normal plasma. The levels in III-3 and IV-5 were at ~90% of pooled normal plasma level (data not shown). As measured by a modified one-stage activated partial thromboplastin time, plasma FXII coagulant activity in four family members (III-1 and IV-3-5), whose blood was collected in sodium citrate, were 1.31, 1.44, 1.28 and 1.21 U/mL, respectively. On immunoblots, plasma FXII protein in all samples had a similar molecular mass, although the level in samples from III-3 and IV-5 was lower (data not shown). No additional FXII protein bands were detected on immunoblots. The data were consistent with the genotype in these individuals. These studies identified an F12 gene mutation in the descendants of the Hageman family. We showed that there was a mutant allele in III-3 and IV-5, which was likely from II-3 who inherited it from I-1 or I-2. It is likely that John Hageman (II-1) also inherited this mutant allele. Given the fact that there was no detectable FXII activity in his blood [2], John Hageman was expected to be either homozygous for this mutant allele or compounded heterozygous with another unknown mutant F12 allele. An inquiry of family history indicated no evidence that his parents were consanguineous but revealed that they emigrated from the central part of Germany, where the same mutant F12 allele was reported in at least five unrelated families [9, 10]. This mutant allele also existed in another unrelated family in Switzerland [10]. All of the individuals with this allele were asymptomatic and identified only by chance during hospital visits. Apparently, this mutation originated for sometime in history and is present in a number of individuals in Europe, especially in Germany. Since no other F12 mutations were found in the descendents of his family, the possibility cannot be excluded that John Hageman may have inherited two copies of the same mutant F12 allele from his parents. The chance encounter of John Hageman by Oscar Ratnoff more than half a century ago had a major role in the history of understanding the blood coagulation system [1, 3, 11–14]. Now FXII is known to be important in the contact activation system to regulate blood pressure, vascular permeability, complement activation, inflammatory responses, angiogenesis, and thrombosis risk [15–17]. Our finding of the F12 gene mutation in the Hageman family, providing an explanation to the long-standing question of John Hageman’s genetic defects, adds a fresh footnote to this important history in hematology.

Prolylcarboxypeptidase (PRCP) is associated with leanness, hypertension, and thrombosis. PRCP-depleted mice have injured vessels with reduced Kruppel-like factor (KLF)2, KLF4, endothelial nitric oxide synthase (eNOS), and thrombomodulin. Does PRCP influence vessel growth, angiogenesis, and injury repair? PRCP depletion reduced endothelial cell growth, whereas transfection of hPRCP cDNA enhanced cell proliferation. Transfection of hPRCP cDNA, or an active site mutant (hPRCPmut) rescued reduced cell growth after PRCP siRNA knockdown. PRCP-depleted cells migrated less on scratch assay and murine PRCP(gt/gt) aortic segments had reduced sprouting. Matrigel plugs in PRCP(gt/gt) mice had reduced hemoglobin content and angiogenic capillaries by platelet endothelial cell adhesion molecule (PECAM) and NG2 immunohistochemistry. Skin wounds on PRCP(gt/gt) mice had delayed closure and reepithelialization with reduced PECAM staining, but increased macrophage infiltration. After limb ischemia, PRCP(gt/gt) mice also had reduced reperfusion of the femoral artery and angiogenesis. On femoral artery wire injury, PRCP(gt/gt) mice had increased neointimal formation, CD45 staining, and Ki-67 expression. Alternatively, combined PRCP(gt/gt) and MRP-14(-/-) mice were protected from wire injury with less neointimal thickening, leukocyte infiltration, and cellular proliferation. PRCP regulates cell growth, angiogenesis, and the response to vascular injury. Combined with its known roles in blood pressure and thrombosis control, PRCP is positioned as a key regulator of vascular homeostasis.

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 PRCPgt/gt mice reveals expression in kidney and vasculature. Invasive telemetric monitorings show that PRCPgt/gt mice have significantly elevated blood pressure. PRCPgt/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 PRCPgt/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 PRCPgt/gt mice with antioxidants (mitoTEMPO, apocynin, Tempol) abrogates the hypertensive, prothrombotic phenotype. Mechanistically, our studies reveal that PRCPgt/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.

Abstract 616 Background: Older investigations suggest that Factor XII (FXII) influences the inflammatory response. FXII deficient patients have reduced leukocyte migration into skin windows. In addition to bradykinin generation, FXII regulates the expression of monocyte FcγII receptor and stimulates monocytes and macrophages to release interleukin (IL)-1 and IL-6. In vitro, purified FXIIa corrects neutrophil aggregation and degranulation defects in FXII-deficient plasma. Our laboratory demonstrated that FXII signals through uPAR, β1 integrin and the EGFR to stimulate ERK1/2 and Akt phosphorylation (Blood 115:5111, 2010). The downstream consequences of this pathway are FXII-induced cell proliferation, growth and angiogenesis. Since FXII modulates wound angiogenesis, we examined the role for FXII in the inflammatory response and wound repair. Investigations: Exon 3 to 8–deleted FXII knockout mice (FXII−/−) were wounded by creating full thickness (5 mm) punch biopsies on their backs. The total surface area of inflammatory cells recruited to the injury site per 20X high power field (HPF) was determined and analyzed by Image J (NIH). We observed that on Day 1, FXII−/− mice exhibit significantly decreased recruitment of CD11 b–labeled inflammatory cells to injury sites compared with wild type mice (WT) [p=0.0136]. Similar results are observed when uPAR KO mice were compared to WT [p=0.0001], suggesting that leukocyte migration in part is mediated through this receptor. Next we determined the nature of cells in the wound sites. FXII−/− mice have reduced wound neutrophils (Gr-1 staining) and macrophages (F4-80 staining). On the thioglycolate (TG)-induced peritoneal inflammation assay, the number of peritoneal exudate cells (PECs) was measured in WT and FXII−/− mice 1 and 7 days following instillation. FXII−/− mice exhibit significantly decreased number of PEC's on days 1 and 7. Giemsa-Wright stain of peritoneal lavage fluid with manual differential counts or flow cytometry shows that there is a disproportionate decrease in neutrophil recruitment in peritoneal fluid of FXII−/− mice. Also, FXII−/− macrophages have reduced adherence to plastic. Identical assays performed on uPAR and bradykinin B2 receptor (B2R) KO mice did not reveal the same inflammatory defects; both uPAR and B2R KO mice have mild decreases in PEC cell numbers following TG instillation, but overall their inflammatory response remained intact. Investigations next determined if the leukocyte migration defect seen in FXII−/− mice is related to plasma FXII or an intrinsic leukocyte defect. On adoptive bone marrow (BM) transplantation experiments, WT bone marrow transplanted to FXII−/− mice corrects the TG-induced PEC migration defect after both 1 and 7 days from instillation. Alternatively, transplantation of FXII−/− bone marrow into WT mice produced a leukocyte migration defect. In order to further discriminate the contribution of FXII in leukocyte migration, we produced plasma FXII deficiency in WT mice using FXII siRNA. WT mice treated with FXII siRNA reduced plasma FXII activity to Conclusions: These data indicate that there is a unique pool of FXII in leukocytes distinct from plasma (hepatic) FXII. Leukocyte FXII, not plasma FXII, is responsible for leukocyte function in wounds and inflammation sites. FXII−/− mice have attenuated wound injury and, paradoxically, improved wound healing rates. Modulation of leukocyte FXII is a target for promotion of wound healing. Disclosures: No relevant conflicts of interest to declare.

Abstract 1142 Background. The S28 serine protease, prolylcarboxypeptidase (PRCP) degrades bradykinin, angiotensin II, alpha melanocyte stimulating hormone and actives plasma prekallikrein. Additionally our studies indicate that PRCP depletions in vivo and in cultured cells are associated with increased reactive oxygen species (ROS) and loss of constitutive anticoagulant function of endothelium (Blood 2011; 117:3929). PRCP-depleted mice are prothrombotic and hypertensive. We observed that PRCP-depleted cells in culture have reduced growth. We posited that PRCP promotes vascular health by influencing cell proliferation, angiogenesis, and wound repair. Methods and Results. Initial investigations determined that PRCP influences vascular endothelial cell proliferation. Bovine aortic endothelial cells (BAEC) were depleted of PRCP by siRNA knockdown resulting in 5% residual mRNA. After transfecting equal numbers of BAEC, at 24 h, the PRCP siRNA transfected cells have reduced proliferation, −18±3 change in cells/high power field (HPF) (mean±SEM), compared to the sham transfected cells, +23±8 cells/HPF, p Conclusions. These combined studies indicate that PRCP levels in endothelial cells influence cell proliferation and growth. In the whole animal this cell biology observation translates into less induced and wound repair angiogenesis. Since PRCP-depleted endothelial cells and vessels from PRCPgt/gt have increased ROS with loss of anticoagulant properties and PRCPgt/gt have higher thrombosis risk, the finding that PRCP also influences endothelial cell growth and angiogenesis suggests that PRCP promotes vascular health and injury repair. Disclosures: No relevant conflicts of interest to declare.

Abstract 700 Background: Bradykinin B2 receptor KO mice (B2R KO) were recognized to have delayed rose bengal and ferric chloride carotid artery thrombosis times and long tail bleeding times. In B2R KO, elevated serum angiotensin II (AngII) binds to an over-expressed angiotensin receptor 2 (AT2R) to increase plasma NO and prostacyclin (Blood 108:192, 2006). It was proposed that the combined presence of elevated AngII and AT2R are essential for the thrombosis protection phenotype. However, after losartan treatment, an angiotensin receptor 1 antagonist, thrombosis remains delayed in B2R KO despite reduction in AngII levels from 258±64 to 40±15 pg/ml. An additional pathway for thrombosis protection in B2R KO mice was sought. Methods and Results: Losartan treatment lowers ACE mRNA in mice along with lowering AngII levels. However, even in the presence of low AngII levels, plasma Ang1-7 levels, the breakdown product of AngII, in losartan-treated B2R KO are increased [21±2.2 pg/ml vs 14.4±0.7 pg/ml in wild type mice (WT)]. Upon examination, B2R KO also have elevated Mas mRNA, the receptor for angiotensin1-7, with over-expressed renal Mas protein. Treatment of B2R KO with A-779, a Mas antagonist, shortens carotid artery thrombosis time (58±4 to 38±4 min) and tail bleeding time (170±13 to 88±8 sec) and lowers plasma concentrations of nitrate (from 21.5±3.6 micromolar in untreated B2R KO to 15±5 micromolar in A-779-treated mice) and 6-keto-PGF1alpha (259±103 pg/ml in untreated B2R KO to 132±58 pg/ml). ADP- or thrombin-induced platelet (plt) activation are not abnormal in B2R KO, either by aggregation studies or by flow cytometric studies examining for fibrinogen binding or JON/A integrin epitope binding and P-Selectin expression, respectively. Although static and flow adherence to collagen was normal, B2R KO plts have defective spreading on collagen (20±0.6 microns in B2R KO versus 27±0.4 microns in WT, p Conclusion: B2R KO are protected from thrombosis by over-expression of two receptors (AT2R and Mas) from the renin-angiotensin system resulting in increased NO and prostacyclin and an acquired platelet function defect. The receptors AT2R and MAS compensate for the absence of the B2R. These studies show novel mechanism(s) whereby receptors in the renin-angiotensin system influence platelet function and arterial thrombosis risk in vivo. Disclosures: No relevant conflicts of interest to declare.

Abstract 368 Background: Factor XII (FXII) participates in inflammation. FXII deficient patients have reduced leukocyte migration into skin windows. Recently, we have determined that FXII signals through uPAR, β1 integrin and the EGFR to stimulate ERK1/2 and Akt phosphorylation (Blood 115:5111, 2010). The downstream consequences of this pathway are FXII-induced cell proliferation and post-natal angiogenesis. Present studies examined if this pathway also influences leukocyte response to injury. Methods: Exon 3- to 8-deleted FXII knockout mice (FXII KO), generously provided by Dr. Frank Castellino, have impaired inflammatory response to injury. FXII KO were wounded by creating full thickness (5 mm) punch biopsies on their backs. The total surface area of inflammatory cells recruited at the injury site/20 × high power field (HPF) was determined and analyzed by Image J (NIH). We observed that FXII KO on Day 1 exhibit significantly decreased recruitment of CD11b-labeled inflammatory cells to injury sites compared with wild type mice (WT) [mean ± SEM, FXII KO: 40220 ± 3732 vs. WT: 59740 ± 6318 total surface area of CD11b positive cells (pixels)/HPF, p=0.0136]. Similar results are observed when uPAR KO were compared to WT mice [39380 ± 5234 vs. 73310 ± 4688, p=0.0001], suggesting that leukocyte migration is mediated through uPAR. On Day 1 of thioglycolate (TG)-induced peritonitis, FXII KO have a mean of 12.62 × 105/mL peritoneal exudate cells (PEC) vs. 25.94 × 105/mL (p=0.0072) observed in WT. Additionally on Day 7, FXII KO have 10.63 × 105/mL PEC vs. WT 25.38 × 105/mL (p< 0.0001). Likewise on Day 7, uPAR KO have 16.34 × 105/mL peritoneal leukocytes recruited vs. WT 25.38 × 105/mL (p=0.0124). These combined studies indicate that FXII influences the degree of leukocyte inflammation and the inflammatory response is partially mediated through uPAR. Studies next determined if leukocyte recruitment promotes wound healing. On Days 3–5 the rate of wound closure of back punch biopsies is faster in FXII KO than WT (p < 0.0327 by one way ANOVA). Again, we observed that FXII KO on Days 2 and 5 exhibit significantly less CD11b-labeled inflammatory cells to the injury site compared to WT (Day 2: p=0.0004, Day 5: p= 0.0075). Leukocyte subpopulation analysis reveals decreased neutrophil migration (Gr-1 positive cells) into the wound area of FXII KO on Day 2 [FXII KO 5669 ± 1844 vs. WT 40490 ± 8564 total surface area of Gr-1 positive cells (pixels)/ HPF, p=0.0018] and Day 5 [FXII KO 6216 ± 3829 vs. WT 34890 ± 7629 total surface area of Gr-1 positive cells (pixels)/HPF, p=0.0176]. Macrophage recruitment into the wound area, as determined by F4-80 antigenicity, increases over time, but remains significantly reduced in FXII KO mice on Day 2 [FXII KO 3111 ± 1115 vs. WT 19140 ± 5767 total surface area of F4-80 positive cells (pixels)/HPF, p=0.0348] and Day 5 [FXII KO 9377 ± 4772 vs. WT 28340 ± 4411 total surface area of F4-80 positive cells (pixels)/HPF, p=0.0171]. These studies indicate that, in FXII deficiency, there is less neutrophil and macrophage-mediated inflammation and this observation correlates with faster wound healing. Finally studies determined if the reduced inflammation seen in FXII KO is the result of host factors or bone marrow-derived cells. Adoptive bone marrow transplant (BMT) experiments were performed where WT or FXII KO bone marrow was transplanted into FXII KO hosts. Eight weeks following the BMT, mice were subjected to TG-induced peritonitis. KO/KO recipients, have significantly decreased number of PEC on day 1 (17.3 × 105/mL ± 3.894 × 105/mL cells) and day 7 (18.6 × 107/mL ± 4.0 × 107/mL cells) when compared to WT/KO recipients [Day 1: 68.25 × 105/mL ± 13.83 × 105/mL cells (p=0.041), Day 7: 190.5 × 107/mL ± 51.80 × 107/mL cells]. These data indicate that FXII interacts with neutrophils and macrophages to promote the inflammatory response; its absence causes decreased inflammatory cell recruitment. Conclusions: Our data indicate that FXII deficiency disrupts the leukocyte response to injury by reducing inflammatory cell recruitment in two murine models. Paradoxically, reduced leukocyte infiltration into skin wounds promotes healing. These investigations indicate a novel role for FXII in inflammation and wound healing and indicate a unique potential target for inflammation therapeutics. Disclosures: No relevant conflicts of interest to declare.

Abstract 3198 Background: Bradykinin B2 receptor KO mice (B2R KO) have delayed rose bengal and ferric chloride carotid artery thrombosis times and long tail bleeding times. In B2R KO mice, elevated serum angiotensin II (AngII) binds to an over-expressed angiotensin receptor 2 (AT2R) to increase plasma NO and PGI2 (Blood 108:192, 2006). The combined presence of elevated AngII and AT2R was essential to have the thrombosis protection phenotype. However, thrombosis remained delayed in B2R KO mice despite reduction in AngII levels from 258+64 to 40+15 pg/ml using losartan, an angiotension 1 receptor antagonist. These data suggested an additional pathway for thrombosis protection in B2R KO mice. Methods and Results: B2R KO mice have elevated bradykinin and bradykinin 1–5 levels with normal thrombin generation times. Losartan treatment lowered ACE mRNA in mice. B2R KO mice also had elevated Mas mRNA, the receptor for angiotensin1-7, with overexpressed renal AT2R and Mas antigens. Treatment of B2R KO with A-779, a Mas antagonist, shortened thrombosis time (58+4 to 38+4 min) and tail bleeding time (170+13 to 88+8 sec) and lowered plasma concentrations of nitrate (21.5+3.6 to 15+5 micromolar) and 6-keto-PGF1alpha (259+103 to 132+58 pg/ml). Immunofluorescent staining and immunoblot demonstrate AT2R and Mas antigen on murine platelets. “Threshold” ADP- and thrombin-induced platelet aggregation was identical for B2R KO and wild type platelets. Platelet activation by arachidonic acid, ionomycin, thrombin, or collagen as measured by flow cytometry (the activated α2bβ3 integrin complex and P-selectin) was similar between B2R KO and wild type platelets. Although static and flow adherence to collagen was normal, B2R KO platelets had defects in spreading on collagen (20+0.6 versus 27+0.4 microns WT, p Conclusion: B2R KO mice are protected from thrombosis by over-expression of two receptors from the renin-angiotensin system that results in increased NO and prostacyclin and an acquired platelet spreading defect. These data suggest that a platelet spreading defect alone protects from thrombosis and indicate that the receptors AT2R and MAS compensate the absence of the B2R. Last, these studies show a novel mechanism(s) by which receptors in the renin-angiotensin system influence platelet function and arterial thrombosis risk in vivo. Disclosures: No relevant conflicts of interest to declare.

Abstract 651 Background. Prolylcarboxypeptidase (PRCP), an S28 serine protease, degrades bradykinin, angiotensin II, and alpha melanocyte stimulating hormone and activates prekallikrein to plasma kallikrein (Blood 103:4554, 2004). In GWAS, it has been recognized as a risk factor for metabolic syndrome, hypertension, and pre-eclampsia. We postulated that PRCP murine hypomorphs (PRCPgt/gt) have a cardiovascular phenotype. Methods and Results. PRCP is mostly found in kidney in proximal tubules. In arteries, it is found both on endothelium and in media. A gene-trap murine hypomorph was created with 7% mRNA and 23% PRCP antigen in renal tissue. Using the Rose Bengal carotid artery thrombosis models, PRCPgt/gt mice had shorter carotid artery occlusion times (24±3 min [mean±SD]) compared to wild type (52±8 min). On a 4% ferric chloride carotid artery thrombosis assay PRCPgt/gt occluded in 21±8 min whereas wild type do not occlude at 60 min. Pharmacologic inhibition of PRCP with Z-pro-prolinal or plasma kallikrein with soybean trypsin inhibitor, Pro-Phe-Arg-Chloromethylketone or PKSI-572 in 3 mouse strains also shortened the time to carotid artery occlusion. PRCPgt/gt were constitutively hypertensive during the late night cycle (122±5 mm Hg mean arterial pressure vs 114±6 mm Hg for wildtype) as measured by carotid artery telemetry. Treatment of these animals with the mitochondria specific antioxidant mitoTEMPO significantly reduced (113±7 mm Hg) the elevated BP. Plasma angiotensin II and bradykinin levels were unaltered in PRCPgt/gt. PRCPgt/gt plasma had a significant increase in contact activation-induced thrombin generation. Aortic and renal reactive oxygen species (ROS) were increased (3.2-fold and 2.8-fold, respectively) in PRCPgt/gt mice as determined by dihydroethidium (DHE) fluorescence. PRCPgt/gt aortic and renal superoxide measured by lucigenin luminescence also was increased (1.6-fold and 1.7-fold, respectively). In PRCPgt/gt kidneys Amplex Red fluorescence, a measure of hydrogen peroxide, was increased 2.4-fold. Renal tissue had 1.6-fold increased uncoupled eNOS on SDS-PAGE. Arterial occlusion times in PRCPgt/gt were corrected by treatment with antioxidant apocynin or tempol. PRCP siRNA knockdowns in HUVEC or mesenchymal embryonic fibroblasts prepared from PRCPgt/gt embryos had increased constitutive DHE fluorescent ROS (2.1-fold and 1.4-fold, respectively). PRCPgt/gt aorta had decreased expression of Kruppel-like factors 2 and 4, thrombomodulin and eNOS. Moreover, PRCP knockdowns in HUVEC had 36% reduced eNOS mRNA expression. Conclusion. These investigations indicate that PRCP is a specific gene/protein target for arterial thrombosis risk and hypertension. Its presence modulates constitutive cell and tissue ROS. Arterial thrombosis risk is related to effect of ROS on endothelial cell anticoagulant mechanisms. Disclosures: No relevant conflicts of interest to declare.