Bloodworks

PATHOGEN-REDUCED, PLASMALYTE-EXTENDED STORED PLATELETS

MOLECULAR AND TRANSLATIONAL STUDIES IN HEMATOLOGIC DISORDERS

VON WILLEBRAND FACTOR IN SICKLE CELL DISEASE PATHOPHYSIOLOGY

BIOLOGIC AND IMMUNOLOGIC ASPECTS OF TRANSFUSION MEDICINE

VON WILLEBRAND FACTOR IN TRAUMATIC BRAIN INJURY AND ASSOCIATED COAGULOPATHY

BIOSYNTHETIC AND FUNCTIONAL CONSEQUENCES OF VON WILLEBRAND DISEASE MUTATIONS

ROLE OF TRPM8 IN COLD-STORED PLATELET TRANSFUSIONS

VWF ACTIVITY: MOLECULAR BIOLOGY, ETHNIC DIVERSITY AND DISEASE ASSOCIATIONS

MOLECULAR AND TRANSLATIONAL STUDIES IN HEMATOLOGIC DISORDERS - THIS APPLICATION FOR AN NHLBI R35 OUTSTANDING INVESTIGATOR AWARD IS TO CONTINUE R35-FUNDED STUDIES ON MECH- ANISM OF DISEASE IN VON WILLEBRAND DISEASE (VWD) AND SICKLE CELL DISEASE (SCD). BOTH DISEASES INVOLVE VON WILLEBRAND FACTOR (VWF). IN VWD, VWF HAS A STARRING ROLE; IN SCD, IT IS ONE OF MANY ACTORS INVOLVED IN A VERY COMPLEX PATHOPHYSIOLOGY. IN TYPES 2A AND 2B VWD, CAUSED BY DYSFUNCTIONAL VWF THAT RESULTS IN LOSS OF THE LARGEST AND HEMOSTATICALLY MOST ACTIVE MULTIMERS, EXCESSIVE ADAMTS13-MEDIATED CLEAVAGE OCCURS AND THE NUMBER OF SMALL, CLEAVED VWF FRAGMENTS IS HIGH. IN THE PROPOSED STUDIES, WE WILL INVESTIGATE THE IMPACT OF THESE FRAGMENTS ON HEMOSTASIS, AS WE HAVE EVIDENCE THAT THE SMALL FRAGMENTS HAVE FUNCTIONS NOT FOUND IN LARGE MULTIMERS, ONE FRAGMENT INTERFERING WITH HEMOSTASIS BY BLOCKING PLATELET BINDING AND VWF SELF-ASSOCIATION, AND THE OTHER COMPETITIVELY INHIBITING ADAMTS13. IN OUR SCD STUDIES, WE HAVE SUBSTANTIAL EVIDENCE THAT OXIDATION- INDUCED CHANGES TO LIPIDS IN THE SICKLE ERYTHROCYTE MEMBRANE AND PLASMA LIPOPROTEINS PLAY MAJOR ROLES IN THE PATHOPHYSIOLOGY OF VASOOCCLUSION. OXIDATION CONVERTS THE SICKLE ERYTHROCYTE TO AN AGONIST CAPABLE OF ACTIVATING PLATELETS, LEUKOCYTES, AND ENDOTHELIAL CELLS, PROBABLY BY GENERATING BIOACTIVE OXIDIZED LIPIDS SUCH AS PLATELET- ACTIVATING FACTOR AND LYSOPHOSPHOLIPIDS. OXIDATION OF USUALLY PROTECTIVE PLASMA HIGH-DENSITY LIPOPROTEIN MAKES IT PROINFLAMMATORY. IN THIS APPLICATION, WE PROPOSE TO USE A WIDE VARIETY OF IN VITRO AND IN VIVO APPROACHES TO ADDRESS THESE QUESTIONS. FOR THE VWD STUDIES, WE WILL STUDY NORMAL BLOOD AND PATIENT BLOOD, RECOMBINANT PRO- TEINS, AND USE AI-ASSISTED STRUCTURE AND DOCKING. EXPERIMENTALLY, WE WILL USE A VARIETY OF MICROFLUIDIC TECHNIQUES AND OBSERVE THE IN VIVO EFFECTS OF VWF CLEAVAGE FRAGMENTS USING INTRAVITAL MICROSCOPY. FOR THE SCD STUDIES, WE WILL CONTINUE TO REFINE MASS SPECTROMETRY TECHNIQUES TO IDENTIFY CANDIDATE OXIDIZED PHOSPHOLIPIDS IN ERYTHRO- CYTES AND LIPOPROTEINS AND STUDY THE LIPIDOMES OF A VARIETY OF PATIENTS TO UNDERSTAND THE EXTENT OF VARIABILITY. FINALLY, WE WILL EVALUATE CANDIDATE LIPIDS FOR THEIR ABILITY TO INDUCE VASOOCCLUSION AND TEST INTERVENTIONS TO RE- DUCE THE NEGATIVE IMPACT OF IN VIVO OXIDATION. WE EXPECT THESE STUDIES TO INCREASE OUR UNDERSTANDING OF THE MECHANISMS OF DISEASE IN THE TWO DISORDERS, TO PROVIDE NEW BIOMARKERS TO STUDY THE DISEASES, AND TO IMPROVE THE TREATMENT IN AFFLICTED PATIENTS, BOTH BY INCREASING UNDERSTANDING AND BY PROVIDING NEW THERAPEUTIC LEADS.

PLATELET TRANSFUSION INDUCED TRANSPLANT REJECTION ACROSS MHA BARRIERS.

PATHOBIOLOGY OF INCOMPATIBLE TRANSFUSION

DISEASE PATHOPHYSIOLOGY IN A MURINE MODEL OF TTP

THE BIOLOGY OF VWF SELF-ASSOCIATION

ULTRA-LARGE VWF AND THROMBOTIC MICROANGIOPATHY

TRANSFUSION INDUCED BMT REJECTION

COLD-STORED PLATELETS TO TREAT BLEEDING IN CARDIAC SURGERY PATIENTS

REGULATION OF VON WILLEBRAND FACTOR REACTIVITY

PREVENTION OF PLATELET ALLOIMMUNIZATION BY COSTIMULATORY BLOCKADE

GENOMIC TYPING OF RED CELL ANTIGENS

PROTEOLYSIS OF ULTRALARGE VON WILLEBRAND FACTOR AND METABOLIC SYNDROME-DIABETES

NOVEL BIOMARKERS PREDICTIVE OF HEPARIN-INDUCED THROMBOCYTOPENIA

UNDERSTANDING THE MECHANISMS OF PLATELET ALLOIMMUNIZATION AND ITS PREVENTION

EXTENDED STORAGE OF PATHOGEN-REDUED PLATELET CONCENTRATES

GENOMIC TYPING OF RED CELL ANTIGENS

A PILOT STUDY OF N-ACETYLCYSTEINE IN SICKLE CELL DISEASE VASO-OCCLUSIVE CRISIS

INFLAMMATION-MEDIATED PLATELET HYPERREACTIVITY AND THROMBOSIS - PROJECT SUMMARY/ABSTRACT THE CANDIDATE IS AN ASSISTANT PROFESSOR IN PEDIATRICS AND A YOUNG PHYSICIAN-SCIENTIST DEDICATED TO DEVELOPING AN ACADEMIC CAREER FOCUSED ON INVESTIGATING THE INTERSECTION OF INFLAMMATION AND THROMBOSIS THROUGH THE STUDY OF THE MECHANISMS BY WHICH INFLAMMATION PROMOTES PLATELET HYPERREACTIVITY. WITH A STRONG BACKGROUND IN INFLAMMATION, MEGAKARYOCYTE, AND PLATELET BIOLOGY, THE CANDIDATE LOOKS TO DEVELOP NEW EXPERTISE IN AUTOPHAGY AND METABOLISM TO DETERMINE THEIR CONTRIBUTION TO PLATELET HYPERREACTIVITY AND THROMBOSIS. THE CAREER DEVELOPMENT PLAN DESCRIBED IN THE PROPOSAL OUTLINES 2 YEARS OF MENTORED TRAINING, INCLUDING TECHNICAL SKILL TRAINING AND CAREER DEVELOPMENT ACTIVITIES, TO PROMOTE THE SUCCESSFUL TRANSITION TO INDEPENDENCE AND FUTURE FUNDING. THE CANDIDATE’S MENTORS AND CO-MENTORS HAVE PROVEN TRACK-RECORDS OF EXCELLENT TRANSLATIONAL RESEARCH PRODUCTIVITY AND SUCCESSFUL MENTORSHIP. THE RESEARCH PLAN: INFLAMMATION CONTRIBUTES TO THE DEVELOPMENT OF CARDIOVASCULAR DISEASE (CVD) AND PROMOTES PLATELET HYPERREACTIVITY AND THROMBOSIS IN OLDER HUMANS (AGING) AND PATIENTS WITH RHEUMATOID ARTHRITIS (RA). THE THROMBOTIC EVENTS EXPERIENCED BY THESE GROUPS ARE CHARACTERIZED BY PLATELET-RICH ARTERIAL CLOTS DENOTING THAT PLATELET HYPERREACTIVITY IS CENTRAL FOR THE ELEVATED MORBIDITY AND MORTALITY IN THESE POPULATIONS. THE CENTRAL HYPOTHESIS OF THIS APPLICATION IS THAT CHRONIC INFLAMMATION PROMOTES PLATELET HYPERREACTIVITY AND THROMBOSIS THROUGH THE REPROGRAMMING OF KEY PLATELET METABOLIC PATHWAYS. THE WORK PROPOSED IN THIS APPLICATION IS SET TO ELUCIDATE THE MECHANISMS BY WHICH CHRONIC INFLAMMATION PROMOTES PLATELET HYPERREACTIVITY THROUGH DYSREGULATION OF CRITICAL METABOLIC REGULATORS OF PLATELET FUNCTION SUCH AS AUTOPHAGY (AIM 1) AND THE PENTOSE PHOSPHATE PATHWAY (PPP, [AIM 2]) IN OLDER HUMANS AND IN PATIENTS WITH RHEUMATOID ARTHRITIS (AIM3). FOR THIS PURPOSE, I HAVE INTEGRATED MULTIDISCIPLINARY MENTORING AND ADVISORY TEAMS OF WORLD-KNOWN EXPERTS IN AUTOPHAGY (ANDREW THORBURN, PH.D.), METABOLOMICS (ANGELO D’ALESSANDRO PH.D.), AND PLATELET BIOLOGY (MATTHEW RONDINA, MD) TO GUIDE THE SUCCESSFUL COMPLETION OF THE PROPOSED WORK. THE MENTORING, SKILLS, AND TOOLS DEVELOPED THROUGHOUT THE K99 PHASE OF THIS AWARD WILL LAY DOWN A SOLID FOUNDATION TO ESTABLISH MY INDEPENDENT RESEARCH PROGRAM FOCUSED ON THE STUDY OF THE PLATELET METABOLIC DETERMINANTS FOR HYPERREACTIVITY AND THROMBOSIS. FINALLY, AND MOST IMPORTANT, DISCOVERIES FROM OUR RESEARCH HAVE THE POTENTIAL TO IMPROVE THE CARE AND OUTCOMES NOT ONLY FOR OLDER INDIVIDUALS AND PATIENTS WITH RA BUT ALSO CHILDREN WITH CHRONIC INFLAMMATORY CONDITIONS SUCH AS JUVENILE IDIOPATHIC ARTHRITIS (JIA) AND SICKLE CELL DISEASE.

METABOLICALLY ABERRANT EXTRACELLULAR MITOCHONDRIA AND OXIDATIVE ENDOTHELIOPATHY AFTER TRAUMATIC BRAIN INJURY - PROJECT SUMMARY HEMOSTASIS IS AN EQUILIBRIUM STATE THAT STOPS BLEEDING AT THE SITE OF VASCULAR INJURY WITHOUT CAUSING SPONTANEOUS AND SYSTEMIC INTRAVASCULAR COAGULATION AND THROMBOSIS. IT CONSISTS OF FIVE DISTINCT BUT INTERCONNECTED COMPONENTS: PLATELETS AND ADHESIVE LIGANDS, COAGULATION, FIBRINOLYSIS, VASCULAR TONE, AND THE ENDOTHELIUM. ALL FIVE COMPONENTS ARE ALTERED DURING TRAUMATIC BRAIN INJURY (TBI), LEADING TO LOCAL AND SYSTEMIC ENDOTHELIOPATHY AND RESULTANT COAGULOPATHY. ENDOTHELIAL INJURIES HAVE BEEN CONSISTENTLY DETECTED DURING TBI AND SERVE AS A KEY CONDUIT TO NOT ONLY HEMOSTATIC DYSREGULATION BUT ALSO VASCULAR LEAKAGE, TISSUE EDEMA, AND INFLAMMATION. HOWEVER, PAST STUDIES PROVIDE LIMITED INSIGHTS INTO THE SUBCELLULAR CHANGES OF EC INJURY, WHICH FORMS THE FOUNDATION OF TBI-INDUCED ENDOTHELIOPATHY. HERE WE PROPOSE TO INVESTIGATE THE MECHANISM OF HOW EXTRACELLULAR MITOCHONDRIA (EXMT) CAUSE EC INJURY DURING ACUTE TBI. OUR PUBLISHED AND PRELIMINARY OBSERVATIONS LED US TO HYPOTHESIZE THAT (1) METABOLICALLY ACTIVE EXMT RELEASED FROM INJURED BRAINS ARE ENDOCYTOSED BY ECS THROUGH THE GTPASE DYNAMIN- AND CD36-MEDIATED PATHWAYS, (2) ENDOCYTOSED EXMT CAUSE THE CYTOPLASMIC RETICULUM TO UNDERGO OXIDATIVE STRESS (ER STRESS) AND TRIGGER UNFOLDED PROTEIN RESPONSE (UPR) WHILE SIMULTANEOUSLY FACILITE MITOCHONDRIAL AUTOPHAGY (MITOPHAGY) TO REMOVE EXMT, AND (3) WHEN UPR AND MITOPHAGY FAIL TO RESTORE ENDOTHELIAL HOMEOSTASIS, ECS UNDERGO APOPTOSIS TO INCREASE PERMEABILITY AND PROCOAGULANT ACTIVITY. THE FORMER CAUSES CEREBRAL EDEMA, HIGH INTRACRANIAL PRESSURE, AND ACUTE LUNG INJURY, WHEREAS THE LATTER CONTRIBUTES TO SYSTEMIC CONSUMPTIVE COAGULATION, THUS WORSENING THE OUTCOME OF TBI. WE WILL TEST THESE HYPOTHESES BY CONDUCTING IN VITRO EXPERIMENTS TO DEFINE EXMT-INDUCED SUBCELLULAR CHANGES, EVALUATING THE EFFICACY OF AGENTS THAT BLOCK THIS EXMT-INDUCED ENDOTHELIOPATHY IN MOUSE MODELS, AND ANALYZING EXMT LEVELS AND OXIDATIVE ACTIVITY IN PLASMA SAMPLES OF TBI PATIENTS TO IDENTIFY PREDICTIVE AND RISK ASSESSMENT MARKERS FOR TBI-INDUCED ENDOTHELIOPATHY. WE HAVE THREE SPECIFIC AIMS. THE SPECIFIC AIM 1 IS TO DEFINE THE PATHWAY OF EXMT-INDUCED ENDOTHELIAL INJURY IN VITRO WITH SPECIFIC FOCUS ON COMMUNICATIONS BETWEEN THE ENDOPLASMIC RETICULUM AND MITOCHONDRIA. THE AIM 2 IS TO EVALUATE THE EFFICACY OF NOVEL AGENTS FOR SPECIFICALLY BLOCKING EXMT-INDUCED ENDOTHELIOPATHY IN MOUSE MODELS. WE PROPOSE TO INVESTIGATE WHETHER BLOCKING EXMT ENDOCYTOSIS OR ACCELERATING THEIR CLEARANCE CAN MITIGATE THE RISK AND REDUCE THE SEVERITY OF ENDOTHELIOPATHY AND COAGULOPATHY IN MICE SUBJECTED TO SEVERE TBI AND IN NON-INJURED MICE INFUSED WITH EXMT. THE SPECIFIC AIM 3 IS TO QUANTIFY LEVELS AND ACTIVITY OF EXMT AND THEIR DERIVATIVES IN PLASMA OF TBI PATIENTS AND EXPLORE THE POSSIBILITY TO USE EXMT RELATED MARKERS FOR CLINICAL RISK ASSESSMENT OF TBI-INDUCED ENDOTHELIAL INJURY. THIS STUDY WILL IDENTIFY NEW PATHWAYS, PREDICTIVE MARKERS, AND THERAPEUTIC TARGETS OF TBI-INDUCED ENDOTHELIOPATHY AND COAGULOPATHY.

TRAUMA AND ACUTE LUNG INJURY: THE ROLE OF RED BLOOD CELL STORAGE AND TRANSFUSION

TRANSFUSION MEDICINE/HEMOSTASIS TRAIL SITE AT PSBC

LMWH FOR POST-PARTUM PROPHYLAXIS IN WOMEN AT RISK OF VENOUS THROMBOSIS

THE BIOLOGY OF VWF SELF-ASSOCIATION

TRAUMATIC BRAIN INJURY-INDUCED COAGULOPATHY

UNDERSTANDING THE PLATELET-CD8+ T CELL INTERACTIONS IN SEPSIS - ABSTRACT SEPSIS REMAINS A SIGNIFICANT GLOBAL HEALTH THREAT DUE TO ITS HIGH MORTALITY RATE AND SUBSTANTIAL ECONOMIC BURDEN. NEARLY HALF OF SEPTIC PATIENTS DEVELOP CHRONIC CRITICAL ILLNESS (CCI), CHARACTERIZED BY PROLONGED ORGAN DYSFUNCTION AND INCREASED SUSCEPTIBILITY TO INFECTIONS. T CELL SUPPRESSION, PARTICULARLY CD8+ T CELL SUPPRESSION, IS A KEY DRIVER OF CCI. LARGE GAPS IN UNDERSTANDING THE PATHOGENESIS OF CCI HAVE HINDERED ITS EARLY IDENTIFICATION AND THE DEVELOPMENT OF EFFECTIVE THERAPIES. OUR RECENT WORK ON PLATELETS DURING SEPSIS REVEALED THAT MAJOR HISTOCOMPATIBILITY COMPLEX CLASS I (MHC-I) ANTIGEN PRESENTATION AND T CELL REGULATING PATHWAYS ARE AMONG THE MOST SIGNIFICANTLY ALTERED PATHWAYS IN PLATELETS. WE FURTHER DISCOVERED THAT PLATELETS ACTIVELY INTERNALIZE, DEGRADE, AND CROSS-PRESENT ANTIGENS VIA MHC-I TO CD8+ T CELLS. USING PLATELET LINEAGE SPECIFIC MHC-I DEFICIENT MICE, WE FOUND THAT PLATELET MHC-I PLAYS A MAJOR ROLE IN CD8+ T CELL SUPPRESSION DURING SEPSIS. THIS RESEARCH PROGRAM FURTHER INVESTIGATES THE CLINICAL IMPACT OF PLATELET MEDIATED CD8+ T CELL SUPPRESSION AND EXPLORES THE MECHANISM OF MHC- I MEDIATED PLATELET–CD8+ T CELL INTERACTIONS IN SEPSIS-ASSOCIATED CCI. THE GOALS FOR OUR LABORATORY'S RESEARCH PROGRAM OVER THE NEXT FIVE YEARS INCLUDE THE FOLLOWING: 1) DETERMINING WHETHER PLATELET–CD8+ T CELL AGGREGATES ARE PRESENT IN SEPSIS PATIENTS AND WHETHER THEY ARE ASSOCIATED WITH ADVERSE OUTCOMES; 2) CHARACTERIZING WHICH CD8+ T CELL SUBSETS ARE INVOLVED AND HOW THEIR FUNCTIONS DIFFER FROM THOSE OF PLATELET-FREE CD8+ T CELLS; AND 3) ELUCIDATING THE STRUCTURE AND MOLECULAR MECHANISMS UNDERLYING THESE PLATELET-T CELL INTERACTIONS AND THEIR IMPACT ON CD8+ T CELL FUNCTION DURING SEPSIS. THIS RESEARCH PROGRAM ADDRESSES A CRITICAL GAP IN OUR UNDERSTANDING OF SEPSIS PATHOGENESIS. BY INVESTIGATING THE PREVIOUSLY UNEXPLORED RELATIONSHIP BETWEEN MHC-I MEDIATED PLATELETS AND CD8+ T CELLS, WE AIM TO PAVE THE WAY FOR IMPROVED LONG- TERM MANAGEMENT STRATEGIES FOR SEPSIS.

OXIDATIVE CYSTEINE MODIFICATION BY THIOL ISOMERASES IN SICKLE CELL DISEASE - PROJECT SUMMARY/ABSTRACT VASO-OCCLUSIVE EVENTS REPRESENT A MAJOR CLINICAL BURDEN IN SICKLE CELL DISEASE (SCD). VASO-OCCLUSIVE EVENTS RECUR IN PATIENTS DESPITE CURRENT TREATMENTS, INCLUDING THE USE OF HYDROXYUREA TO INCREASE FETAL HEMOGLOBIN AND CRIZANLIZUMAB THAT TARGETS P-SELECTIN FOR CELLULAR ADHESION. OXIDATIVE STRESS IN SCD INCREASES THE RISK FOR VASO- OCCLUSION AND CURRENT ANTI-OXIDATIVE TREATMENTS, INCLUDING L-GLUTAMINE, SHOW EFFICACY IN DECREASING THESE EVENTS. HOWEVER, ANTIOXIDANTS DO NOT AMELIORATE VASO-OCCLUSIVE CRISES. NEW TREATMENT STRATEGIES FOR VASO-OCCLUSION IN SICKLE CELL DISEASE BASED ON AN IMPROVED UNDERSTANDING OF THE REDOX MECHANISMS ARE REQUIRED. THIOL ISOMERASES BELONG TO A CLASS OF OXIDOREDUCTASES THAT ARE SECRETED FROM PLATELETS AND ENDOTHELIAL CELLS AND ARE REQUIRED FOR THROMBUS FORMATION. THE ARCHETYPAL THIOL ISOMERASE, PROTEIN DISULFIDE ISOMERASE (PDI), PROMOTES THROMBOINFLAMMATION IN SCD, IS SENSITIVE TO THE REDOX ENVIRONMENT, AND CAN BE TARGETED WITH FLAVONOIDS SUCH AS ISOQUERCETIN. OUR PRELIMINARY DATA THAT ISOQUERCETIN DECREASES CELL-CELL ADHESION IN SCD MICE SUGGESTS THAT PDI COULD BE A POTENTIAL TARGET FOR VASO-OCCLUSION. HOWEVER, THE MECHANISM BY WHICH PDI PROMOTES VASO- OCCLUSION IS UNCLEAR. THIS PROPOSAL WILL TEST THE CENTRAL HYPOTHESIS THAT THIOL ISOMERASES PROMOTE REDOX-SENSITIVE VASO-OCCLUSION THROUGH CYSTEINE ELECTRON TRANSFERRING EVENTS IN SICKLE CELL DISEASE. WE WILL EVALUATE REDOX STRESS- MEDIATED VASO-OCCLUSION IN SCD IN THREE INTEGRATED AIMS USING CELL AND CHEMICAL BIOLOGY APPROACHES WITH MURINE MODELS OF THE DISEASE. IN AIM 1, WE WILL MECHANISTICALLY EXAMINE THE CAPACITY OF PDI TO SENSE THE REDOX ENVIRONMENT IN SCD TO PROMOTE ELECTRON TRANSFERS IN THE FORM OF CYSTEINE DISULFIDES. THIS AIM WILL DETERMINE WHETHER REDUCED OR OXIDIZED PDI PROMOTES PLATELET AND NEUTROPHIL ACTIVATION IN SCD BY CATALYZING ELECTRON WITHDRAWAL FROM THEIR KNOWN REDOX TARGETS. IN AIM 2, WE WILL TRANSITION OUR STUDIES TO EVALUATE THE FUNCTION OF ELECTRON WITHDRAWAL MECHANISMS CATALYZE BY THIOL ISOMERASES USING INTRAVITAL MICROSCOPY TO OBSERVE THROMBOSIS, HEMOSTASIS, AND VASO-OCCLUSION IN SCD. WE WILL ALSO COMPLEMENT THE STUDIES BY OBSERVING THE FUNCTION OF THIOL ISOMERASE-MEDIATED ELECTRON WITHDRAWAL ON LEUKOCYTE CELL-CELL ADHESION EVENTS IN VIVO. LASTLY, AIM 3 WILL UTILIZE CARBON NUCLEOPHILIC PROBES THAT TAG SPECIFIC CYSTEINE SULFUR OXOFORMS TO PROBE THE GLOBAL FUNCTION OF ELECTRON TRANSFERRING EVENTS IN SCD. THE PROBES WILL IDENTIFY NEW TARGETS OF THIOL ISOMERASES IN AN UNBIASED MANNER IN ORDER TO DETERMINE WHETHER A CHARACTERISTIC SET OF CYSTEINE DISULFIDE SCISSION OR FORMATION IS REQUIRED FOR VASO - OCCLUSION. THE PROBES WILL ALSO IDENTIFY MECHANISTICALLY THE ROLE OF CYSTEINE ELECTRON TRANSFERRING EVENTS ON HEMOGLOBIN FUNCTION FOR RED BLOOD CELL SICKLING AND LEUKOCYTE-MEDIATED CELL-CELL ADHESION FOR VASO-OCCLUSIVE EVENTS. THE K99 PHASE WILL FOCUS ON AIMS 1 AND 2 WHEREAS THE R00 PHASE WILL FOCUS ON AIM 3. THE ADDITIONAL TRAINING AFFORDED BY THIS CAREER DEVELOPMENT AWARD WILL NOT ONLY ENABLE ME TO EXPAND MY SKILLSETS, BUT WILL ALSO UNIQUELY POSITION ME TO BUILD AN INDEPENDENT RESEARCH PROGRAM FOCUSED ON OXIDATIVE CYSTEINE MODIFICATION IN THE REDOX-REGULATED VASO-OCCLUSIVE EVENTS OF SCD.

INFLAMMATION-ASSOCIATED AUTOPHAGY DYSFUNCTION - PROJECT SUMMARY CHRONIC TNF-DRIVEN INFLAMMATION AND DEREGULATED HEMOSTASIS ARE CHARACTERISTIC OF SICKLE CELL DISEASE (SCD). DESPITE TNFΑ'S PIVOTAL ROLE IN THE PATHOPHYSIOLOGY OF SCD AND ITS ROLE IN DEREGULATING HEMOSTASIS IN OTHER INFLAMMATORY DISEASES, THE PRECISE MECHANISMS BY WHICH IT DISRUPTS THE HEMOSTATIC BALANCE IN SCD REMAIN POORLY UNDERSTOOD. IN RESPONSE TO TNFΑ, PLATELETS ACCUMULATE LARGER AMOUNTS OF MITOCHONDRIA. SUCH MODIFICATION IS PARALLELED BY FUNCTIONAL CHANGES THAT DEREGULATE PLATELETS' HEMOSTATIC ROLE. MORE RECENTLY, IT HAS BEEN DEMONSTRATED THAT IN SCD, A PROPORTION OF MATURE RED BLOOD CELLS (RBCS) CONTAIN DYSFUNCTIONAL MITOCHONDRIA THAT PROMOTE HEMOLYSIS. GIVEN THAT BOTH RBCS AND PLATELETS ORIGINATE FROM COMMON HEMATOPOIETIC PRECURSORS (MEGAKARYOCYTIC ERYTHROID PROGENITORS), TNFΑ MAY DISRUPT COMMON REGULATORY PATHWAYS IN PLATELETS AND ERYTHROID CELLS RESPONSIBLE FOR THE CLEARANCE OF MITOCHONDRIA. HENCE, WE HYPOTHESIZE THAT TNF-DRIVEN INFLAMMATION IN SCD BLOCKS AUTOPHAGY AND MITOPHAGY IN PLATELETS AND RETICULOCYTES, 1) LEADING TO THE ACCUMULATION OF DYSFUNCTIONAL MITOCHONDRIA AND DEREGULATION OF PLATELET’S HEMOSTATIC FUNCTION AND 2) DISRUPTING THE CLEARANCE OF MITOCHONDRIA BY RETICULOCYTES THAT CONSEQUENTLY GENERATE MATURE RBCS WITH RETAINED MITOCHONDRIA. OUR LONG- TERM GOAL IS TO IDENTIFY INFLAMMATION-MEDIATED MECHANISMS THAT DISRUPT AUTOPHAGY IN PLATELETS AND ERYTHROID CELLS IN SCD. ULTIMATELY, WE AIM TO DEVELOP NOVEL THERAPIES TO EFFECTIVELY ADDRESS THESE PATHOPHYSIOLOGICAL ASPECTS OF THE DISEASE.

MASTL KINASE ACTIVITY IN MEGAKARYOCYTE DIFFERENTIATION

NATURAL VARIATION IN VWF AND FVIII

OXIDANT REGULATION OF PROTEOLYSIS IN ACUTE LUNG INJURY