Michael A. Bukys

Notch-mediated patterning and cell fate allocation of pancreatic progenitor cells

Early pancreatic morphogenesis is characterized by the transformation of an uncommitted pool of pancreatic progenitor cells into a branched pancreatic epithelium that consists of ‘tip’ and ‘trunk’ domains. These domains have distinct molecular signatures and differentiate into distinct pancreatic cell lineages. Cells at the branched tips of the epithelium develop into acinar cells, whereas cells in the trunk subcompartment differentiate into endocrine and duct cells. Recent genetic analyses have highlighted the role of key transcriptional regulators in the specification of these subcompartments. Here, we analyzed in mice the role of Notch signaling in the patterning of multipotent pancreatic progenitor cells through mosaic overexpression of a Notch signaling antagonist, dominant-negative mastermind-like 1, resulting in a mixture of wild-type and Notch-suppressed pancreatic progenitor cells. We find that attenuation of Notch signaling has pronounced patterning effects on multipotent pancreatic progenitor cells prior to terminal differentiation. Relative to the wild-type cells, the Notch-suppressed cells lose trunk marker genes and gain expression of tip marker genes. The Notch-suppressed cells subsequently differentiate into acinar cells, whereas duct and endocrine populations are formed predominantly from the wild-type cells. Mechanistically, these observations could be explained by a requirement of Notch for the expression of the trunk determination gene Nkx6.1. This was supported by the finding of direct binding of RBP-jκ to the Nkx6.1 proximal promoter.

The contribution of amino acid region Asp695-Tyr698 of factor V to procofactor activation and factor Va function

There is strong evidence that a functionally important cluster of amino acids is located on the COOH-terminal portion of the heavy chain of factor Va, between amino acid residues 680 and 709. To ascertain the importance of this region for cofactor activity, we have synthesized five overlapping peptides representing this amino acid stretch (10 amino acids each, HC1-HC5) and tested them for inhibition of prothrombinase assembly and function. Two peptides, HC3 (spanning amino acid region 690-699) and HC4 (containing amino acid residues 695-704), were found to be potent inhibitors of prothrombinase activity with IC50 values of ∼12 and ∼10 μm, respectively. The two peptides were unable to interfere with the binding of factor Va to active site fluorescently labeled Glu-Gly-Arg human factor Xa, and kinetic analyses showed that HC3 and HC4 are competitive inhibitors of prothrombinase with respect to prothrombin with Ki values of ∼6.3 and ∼5.3 μm, respectively. These data suggest that the peptides inhibit prothrombinase because they interfere with the incorporation of prothrombin into prothrombinase. The shared amino acid motif between HC3 and HC4 is composed of Asp695-Tyr-Asp-Tyr-Gln699 (DYDYQ). A pentapeptide with this sequence inhibited both prothrombinase function with an IC50 of 1.6 μm (with a KD for prothrombin of 850 nm), and activation of factor V by thrombin. Peptides HC3, HC4, and DYDYQ were also found to interact with immobilized thrombin. A recombinant factor V molecule with the mutations Asp695 → Lys, Tyr696 → Phe, Asp697 → Lys, and Tyr698 → Phe (factor V2K2F) was partially resistant to activation by thrombin but could be readily activated by RVV-V activator (factor VaRVV2K2F) and factor Xa (factor VaXa2K2F). Factor VaRVV2K2F and factor VaXa2K2F had impaired cofactor activity within prothrombinase in a system using purified reagents. Our data demonstrate for the first time that amino acid sequence 695-698 of factor Va heavy chain is important for procofactor activation and is required for optimum prothrombinase function. These data provide functional evidence for an essential and productive contribution of factor Va to the activity of prothrombinase.

Notch-mediated post-translational control of Ngn3 protein stability regulates pancreatic patterning and cell fate commitment.

Ngn3 is recognized as a regulator of pancreatic endocrine formation, and Notch signaling as an important negative regulator Ngn3 gene expression. By conditionally controlling expression of Ngn3 in the pancreas, we find that these two signaling components are dynamically linked. This connection involves transcriptional repression as previously shown, but also incorporates a novel post-translational mechanism. In addition to its ability to promote endocrine fate, we provide evidence of a competing ability of Ngn3 in the patterning of multipotent progenitor cells in turn controlling the formation of ducts. On one hand, Ngn3 cell-intrinsically activates endocrine target genes; on the other, Ngn3 cell-extrinsically promotes lateral signaling via the Dll1>Notch>Hes1 pathway which substantially limits its ability to sustain endocrine formation. Prior to endocrine commitment, the Ngn3-mediated activation of the Notch>Hes1 pathway impacts formation of the trunk domain in the pancreas causing multipotent progenitors to lose acinar, while gaining endocrine and ductal, competence. The subsequent selection of fate from such bipotential progenitors is then governed by lateral inhibition, where Notch>Hes1-mediated Ngn3 protein destabilization serves to limit endocrine differentiation by reducing cellular levels of Ngn3. This system thus allows for rapid dynamic changes between opposing bHLH proteins in cells approaching a terminal differentiation event. Inhibition of Notch signaling leads to Ngn3 protein stabilization in the normal mouse pancreas explants. We conclude that the mutually exclusive expression pattern of Ngn3/Hes1 proteins in the mammalian pancreas is partially controlled through Notch-mediated post-translational regulation and we demonstrate that the formation of insulin-producing beta-cells can be significantly enhanced upon induction of a pro-endocrine drive combined with the inhibition of Notch processing.

The Structural Integrity of Anion Binding Exosite I of Thrombin Is Required and Sufficient for Timely Cleavage and Activation of Factor V and Factor VIII

α-Thrombin has two separate electropositive binding exosites (anion binding exosite I, ABE-I and anion binding exosite II, ABE-II) that are involved in substrate tethering necessary for efficient catalysis. α-Thrombin catalyzes the activation of factor V and factor VIII following discrete proteolytic cleavages. Requirement for both anion binding exosites of the enzyme has been suggested for the activation of both procofactors by α-thrombin. We have used plasma-derived α-thrombin, β-thrombin (a thrombin molecule that has only ABE-II available), and a recombinant prothrombin molecule rMZ-II (R155A/R284A/R271A) that can only be cleaved at Arg320 (resulting in an enzymatically active molecule that has only ABE-I exposed, rMZ-IIa) to ascertain the role of each exosite for procofactor activation. We have also employed a synthetic sulfated pentapeptide (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{DY}(\mathrm{SO}_{3}^{-})\mathrm{DY}(\mathrm{SO}_{3}^{-})\mathrm{Q}\) \end{document}, designated D5Q1,2) as an exosite-directed inhibitor of thrombin. The clotting time obtained with β-thrombin was increased by ∼8-fold, whereas rMZ-IIa was 4-fold less efficient in promoting clotting than α-thrombin under similar experimental conditions. α-Thrombin readily activated factor V following cleavages at Arg709, Arg1018, and Arg1545 and factor VIII following proteolysis at Arg372, Arg740, and Arg1689. Cleavage of both procofactors byα-thrombin was significantly inhibited by D5Q1,2. In contrast, β-thrombin was unable to cleave factor V at Arg1545 and factor VIII at both Arg372 and Arg1689. The former is required for light chain formation and expression of optimum factor Va cofactor activity, whereas the latter two cleavages are a prerequisite for expression of factor VIIIa cofactor activity. β-Thrombin was found to cleave factor V at Arg709 and factor VIII at Arg740, albeit less efficiently than α-thrombin. The sulfated pentapeptide inhibited moderately both cleavages by β-thrombin. Under similar experimental conditions, membrane-bound rMZ-IIa cleaved and activated both procofactor molecules. Activation of the two procofactors by membrane-bound rMZ-IIa was severely impaired by D5Q1,2. Overall the data demonstrate that ABE-I alone of α-thrombin can account for the interaction of both procofactors with α-thrombin resulting in their timely and efficient activation. Because formation of meizothrombin precedes that of α-thrombin, our findings also imply that meizothrombin may be the physiological activator of both procofactors in vivo in the presence of a procoagulant membrane surface during the early stages of coagulation.

A Control Switch for Prothrombinase CHARACTERIZATION OF A HIRUDIN-LIKE PENTAPEPTIDE FROM THE COOH TERMINUS OF FACTOR Va HEAVY CHAIN THAT REGULATES THE RATE AND PATHWAY FOR PROTHROMBIN ACTIVATION

Membrane-bound factor Xa alone catalyzes prothrombin activation following initial cleavage at Arg271 and prethrombin 2 formation (pre2 pathway). Factor Va directs prothrombin activation by factor Xa through the meizothrombin pathway, characterized by initial cleavage at Arg320 (meizo pathway). We have shown previously that a pentapeptide encompassing amino acid sequence 695–699 from the COOH terminus of the heavy chain of factor Va (Asp-Tyr-Asp-Tyr-Gln, DYDYQ) inhibits prothrombin activation by prothrombinase in a competitive manner with respect to substrate. To understand the mechanism of inhibition of thrombin formation by DYDYQ, we have studied prothrombin activation by gel electrophoresis. Titration of plasma-derived prothrombin activation by prothrombinase, with increasing concentrations of peptide, resulted in complete inhibition of the meizo pathway. However, thrombin formation still occurred through the pre2 pathway. These data demonstrate that the peptide preferentially inhibits initial cleavage of prothrombin by prothrombinase at Arg320. These findings were corroborated by studying the activation of recombinant mutant prothrombin molecules rMZ-II (R155A/R284A/R271A) and rP2-II (R155A/R284A/R320A) which can be only cleaved at Arg320 and Arg271, respectively. Cleavage of rMZ-II by prothrombinase was completely inhibited by low concentrations of DYDYQ, whereas high concentrations of pentapeptide were required to inhibit cleavage of rP2-II. The pentapeptide also interfered with prothrombin cleavage by membrane-bound factor Xa alone in the absence of factor Va increasing the rate for cleavage at Arg271 of plasma-derived prothrombin or rP2-II. Our data demonstrate that pentapeptide DYDYQ has opposing effects on membrane-bound factor Xa for prothrombin cleavage, depending on the incorporation of factor Va in prothrombinase.

The interaction of fragment 1 of prothrombin with the membrane surface is a prerequisite for optimum expression of factor Va cofactor activity within prothrombinase

Incorporation of factor (F) Va into prothrombinase directs prothrombin activation by FXa through the meizothrombin pathway, characterized by initial cleavage at Arg(320). We have shown that a pentapeptide with the sequence DYDYQ specifically inhibits this pathway. It has been also established that Hir(54-65)(SO(3)(-)) is a specific inhibitor of prothrombinase. To understand the role of FVa within prothrombinase at the molecular level, we have studied thrombin formation by prothrombinase in the presence of various prothrombin-derived fragments alone or in combination. Activation of prethrombin 1 is slow with cleavages at Arg(320) and Arg(271) occurring with similar rates. Addition of purified fragment 1 to prethrombin 1 accelerates both the rate of cleavage at Arg(320) and thrombin formation. Both reactions were inhibited by Hir(54-65)(SO(3)(-)) while DYDYQ had no significant inhibitory effect on prethrombin 1 cleavage in the absence or presence of fragment 1. Similarly, activation of prethrombin 2 by prothrombinase, is inhibited by Hir(54-65)(SO(3)(-)), but is not affected by DYDYQ. Addition of purified fragment 1*2 to prethrombin 2 accelerates the rate of cleavage at Arg(320) by prothrombinase. This addition also results in a significant inhibition of thrombin formation by DYDYQ and is concurrent with the elimination of the inhibitory effect of Hir(54-65)(SO(3)(-)) on the same reaction. Finally, a membrane-bound ternary complex composed of prethrombin 2/fragment 1*2/Hir(54-65)(SO(3)(-)) is inhibited by DYDYQ. Altogether, the data demonstrate that membrane-bound fragment 1 is required to promote optimum Fva cofactor activity which in turn is translated by efficient initial cleavage of prothrombin by prothrombinase at Arg(320).

Role of the acidic hirudin-like COOH-terminal amino acid region of factor Va heavy chain in the enhanced function of prothrombinase.

Prothrombinase activates prothrombin through initial cleavage at Arg320 followed by cleavage at Arg271. This pathway is characterized by the generation of an enzymatically active, transient intermediate, meizothrombin, that has increased chromogenic substrate activity but poor clotting activity. The heavy chain of factor Va contains an acidic region at the COOH terminus (residues 680−709). We have shown that a pentapeptide from this region (DYDYQ) inhibits prothrombin activation by prothrombinase by inhibiting meizothrombin generation. To ascertain the function of these regions, we have created a mutant recombinant factor V molecule that is missing the last 30 amino acids from the heavy chain (factor VΔ680−709) and a mutant molecule with the 695DYDY698 → AAAA substitutions (factor V4A). The clotting activities of both recombinant mutant factor Va molecules were impaired compared to the clotting activity of wild-type factor Va (factor VaWt). Using an assay employing purified reagents, we found that prothrombinase assembled with factor VaΔ680−709 displayed an ∼39% increase in kcat, while prothrombinase assembled with factor Va4A exhibited an ∼20% increase in kcat for the activation of prothrombin as compared to prothrombinase assembled with factor VaWt. Gel electrophoresis analyzing prothrombin activation by prothrombinase assembled with the mutant molecules revealed a delay in prothrombin activation with persistence of meizothrombin. Our data demonstrate that the COOH-terminal region of factor Va heavy chain is indeed crucial for coordinated prothrombin activation by prothrombinase because it regulates meizothrombin cleavage at Arg271 and suggest that this portion of factor Va is partially responsible for the enhanced procoagulant function of prothrombinase.

Contribution of Amino Acid Region 334−335 from Factor Va Heavy Chain to the Catalytic Efficiency of Prothrombinase

We have demonstrated that amino acids E323, Y324, E330, and V331 from the factor Va heavy chain are required for the interaction of the cofactor with factor Xa and optimum rates of prothrombin cleavage. We have also shown that amino acid region 332−336 contains residues that are important for cofactor function. Using overlapping peptides, we identified amino acids D334 and Y335 as contributors to cofactor activity. We constructed recombinant factor V molecules with the mutations D334 → K and Y335 → F (factor VKF) and D334 → A and Y335 → A (factor VAA). Kinetic studies showed that while factor VaKF and factor VaAA had a KD for factor Xa similar to the KD observed for wild-type factor Va (factor VaWT), the clotting activities of the mutant molecules were impaired and the kcat of prothrombinase assembled with factor VaKF and factor VaAA was reduced. The second-order rate constant of prothrombinase assembled with factor VaKF or factor VaAA for prothrombin activation was ∼10-fold lower than the second-order rate constant for the same reaction catalyzed by prothrombinase assembled with factor VaWT. We also created quadruple mutants combining mutations in the amino acid region 334–335 with mutations at the previously identified amino acids that are important for factor Xa binding (i.e., E323Y324 and E330V331). Prothrombinase assembled with the quadruple mutant molecules displayed a second-order rate constant up to 400-fold lower than the values obtained with prothrombinase assembled with factor VaWT. The data demonstrate that amino acid region 334–335 is required for the rearrangement of enzyme and substrate necessary for efficient catalysis of prothrombin by prothrombinase.

Cooperative regulation of the activity of factor Xa within prothrombinase by discrete amino acid regions from factor Va heavy chain.

The prothrombinase complex catalyzes the activation of prothrombin to α-thrombin. We have repetitively shown that amino acid region 695DYDY698 from the COOH terminus of the heavy chain of factor Va regulates the rate of cleavage of prothrombin at Arg271 by prothrombinase. We have also recently demonstrated that amino acid region 334DY335 is required for the optimal activity of prothrombinase. To assess the effect of these six amino acid residues on cofactor activity, we created recombinant factor Va molecules combining mutations at amino acid regions 334–335 and 695−698 as follows: factor V3K (334DY335 → KF and 695DYDY698 → KFKF), factor VKF/4A (334DY335 → KF and 695DYDY698 → AAAA), and factor V6A (334DY335 → AA and 695DYDY698 → AAAA). The recombinant factor V molecules were expressed and purified to homogeneity. Factor Va3K, factor VaK4/4A, and factor Va6A had reduced affinity for factor Xa, when compared to the affinity of the wild-type molecule (factor VaWt) for the enzyme. Prothrombinase assembled with saturating concentrations of factor Va3K had a 6-fold reduced second-order rate constant for prothrombin activation compared to the value obtained with prothrombinase assembled with factor VaWt, while prothrombinase assembled with saturating concentrations of factor VaKF/4A and factor Va6A had approximately 1.5-fold reduced second-order rate constants. Overall, the data demonstrate that amino acid region 334–335 together with amino acid region 695−698 from factor Va heavy chain are part of a cooperative mechanism within prothrombinase regulating cleavage and activation of prothrombin by factor Xa.

Identification of an inactivating cleavage site for α-thrombin on the heavy chain of factor Va

Previous studies of factor (F)Va inactivation on human umbilical vein endothelial cells have shown that α-thrombin cleaves the heavy chain near the COOH-terminus to produce a Mr 97,000 fragment containing the NH2-terminal portion of the heavy chain and a Mr 8,000 peptide containing the rest of the molecule. The α-thrombin cleavage appeared to occur between amino acid residues 586 and 654 of FV.This region contains a consensus sequence for α-thrombin cleavage located at residues 640–644 (S-S-P-R-S). To test the hypothesis that α-thrombin cleaves the FVa heavy chain at Arg643 and to evaluate the functional importance of this cleavage for FVa inactivation, sitedirected mutagenesis was used to create recombinant FV molecules with mutations R643→Q (FVR643Q) and R643→A (FVR643A). All recombinant molecules were purified to homogeneity and assayed for activity following extended activation with α-thrombin. Under similar experimental conditions, appearance of the Mr 97,000 heavy chain fragment in the plasma and wild-type FVa molecules correlated with partial loss of cofactor activity, while following extended incubation of FVR643Q and FVR643A with α-thrombin no cleavage of the heavy chain at Arg643 was detected and no presence of the Mr 97,000 heavy-chain fragment was noticed. Further, no loss in cofactor activity was observed using these mutant recombinant FVa molecules. Our data demonstrate that cleavage of FVa at Arg643 by α-thrombin results in a partially inactive cofactor molecule and provides for an activated protein C (APC)-independent anticoagulant effect of α-thrombin.