Ambivalent professional identity of early remedial medical students from Generation Z: a qualitative study.

BACKGROUND: Supporting professional identity development in medical students undergoing remediation in the first few years of their studies is an important topic. However, there is a lack of research on developing an effective and individualised process for successful remediation that targets learner identities. This study examined the identities of Generation Z remedial medical students through the lens of professional identity formation, focusing on the difficulties they faced and the support they sought. METHODS: An exploratory qualitative case study was conducted within a constructivist paradigm. Twenty-two medical students (14 males and 8 females) who had experienced remediation in their first few years of medical university participated in this study. All participants were members of Generation Z. Qualitative data were collected through face-to-face, semi-structured interviews and analysed using thematic analysis. RESULTS: Medical students undergoing remediation in the first few years experienced resistance to the medical profession and conflict due to the gap between the ideal and the reality they experienced after entering medical university. Students' professional identities were closely intertwined with their pre-university identities; this affected the process of professional identity formation after entering medical university. They preferred assurances of confidentiality as a prerequisite and immediately sought advice through social networks to support their professional identity development. CONCLUSIONS: When planning professional identity development support for Generation Z medical students undergoing remediation in the first few years, it is necessary to carefully select integrative interaction methods, focus on the context of individual learners, and collaboratively discuss specific responses between students and faculty. The results of this study could be useful to faculty in developing support systems for future remedial medical students that focuses on professional identity development and mentoring of remedial medical students.

Flexible e-learning video approach to improve fundus examination skills for medical students: a mixed-methods study.

BACKGROUND: Training for the fundus examination using traditional teaching is challenging, resulting in low generalist physicians' confidence in performing the funduscopic examination. There is growing evidence suggesting a flexible e-learning video approach's value in teaching physical examination procedures. However, whether the flexible e-learning video approach is superior to the traditional, face-to-face (F2F) lecture-based teaching for the funduscopic exam and the cognitive processes supporting its effectiveness has not yet been determined. METHODS: We conducted a sequential explanatory mixed-method study to compare the flexible e-learning video approach's effectiveness versus the F2F lecture-based approach for teaching the funduscopic exam to medical students at Chiba University in Japan. Medical students were randomly assigned to either a flexible e-learning video approach group or a F2F lecture approach group. We then quantitatively measured the diagnostic accuracy of funduscopic findings before and after attending the specific classrooms. Next, we conducted student focus groups to explore the students' thinking processes in the flexible e-learning video approach vs. the F2F lecture-based teaching of fundus examination. The qualitative data were analyzed using the qualitative content analysis method. RESULTS: The mean diagnostic accuracy scores in the post-test significantly increased from pre-test in the intervention group (36.6 to 63.4%, p < 0.001). Post-post comparisons across the two groups revealed a significant difference (intervention group 63.4% vs. control group 34.6%, p < 0.001). Six semi-structured focused group interviews were conducted (n = 36). In the flexible e-learning video approach group, we identified ten categories corresponding to four levels of the revised Bloom's taxonomy: remember, understand, apply, analyze. Five categories were identified in the traditional F2F lecture approach group corresponding to three revised Bloom's taxonomy levels: understand, apply, analyze. Interrater reliability was substantial (Cohen's kappa = 0.81). CONCLUSIONS: Teaching medical students funduscopic examination using the flexible e-learning video approach leads to improved diagnostic accuracy of funduscopic examinations. The flexible e-learning video teaching method enabled higher cognitive activity levels than the traditional, lecture-based classroom, as assessed using the revised Bloom's taxonomy. TRIAL REGISTRATION: This study was registered with the University Hospital Medical Information Network Clinical Trials Registry on 08/02/2020 (Unique trial number: UMIN 000039434 ).

Improved cognitive apprenticeship clinical teaching after a faculty development program.

BACKGROUND: While it is well known that the cognitive apprenticeship is an effective workplace-based teaching approach for clinical teachers, the effects of faculty development (FD) have not been analyzed from that perspective. The purpose of this study was to investigate self-assessment by clinical teachers of their educational perceptions and behaviors after a FD program using the cognitive apprenticeship model. METHODS: Board-certified pediatricians who participated in a 3-day FD program on practical clinical teaching were asked to complete questionnaires. Fifty participants completed two questionnaires prior to and 3 and 6 months after the FD program: the first was on the participants' general perceptions and behaviors in relation to their own clinical education and the second was a self-assessment using the Maastricht Clinical Teaching Questionnaire (MCTQ) that was developed based on the cognitive apprenticeship model. RESULTS: The general survey demonstrated that 78% of the participants experienced positive changes in their educational perceptions 6 months after FD. Self-assessment using the MCTQ showed that the scores in the categories of "articulation," "exploration," and "safe learning environment" remained significantly improved 6 months after the FD program. CONCLUSIONS: The participants' self-perceived improvement in behaviors was sustainable for 6 months after participation the FD program. The results of the MCTQ show that through their experiences in the FD program, the participants seemingly transformed their clinical teaching to become interactive facilitators, encouraging self-directed learning. Our results also suggest that the MCTQ can be used for self-assessment of clinical teachers and to enhance the effectiveness of the FD program.

Good clinical teachers in pediatrics: The perspective of pediatricians in Japan.

BACKGROUND: The purpose of this study was to identify the attributes of good clinical teachers in pediatrics (CTPs) in Japan, and to elucidate pediatricians and pediatric trainees' perceptions of these attributes. METHODS: The attributes of good CTPs were identified qualitatively by conducting a thematic analysis of questionnaires answered by board-certified pediatricians and pediatric trainees. The attributes identified were rated quantitatively by a large number of participants in both groups. RESULTS: Forty-eight individual attributes of good CTPs were identified, which were classified into three domains: personal, pediatrician, and teacher. The three domains and most of the attributes were consistent with previous studies. However, a few additional attributes, including "is kind/thoughtful toward others" and "defends trainees", which may be unique to pediatricians in Japan, were identified. Significant differences in the pediatricians' and trainees' perceptions of these attributes were elucidated: The differences were most noticeable for teacher attributes and least for personal attributes. CONCLUSION: Although most of the identified attributes of good CTPs in our study appear to be universal, there were significant differences in the pediatricians' and trainees' perceptions of good CTPs, especially in relation to teacher attributes. Our study provides additional bases for good CTPs and future faculty development, for enhanced pediatric clinical education.

Launch of Board Certification in Pediatric Infectious Diseases in Japan.

To cultivate specialists in pediatric infectious diseases (ID) in Japan, the Japanese Society for Pediatric Infectious Diseases initiated board certification for pediatric ID in 2017. Previously, in 2014, we had formed a committee for board certification in pediatric ID and discussed the fundamentals of the board certification system, including the goals, requirements for designated training institutions, provisional certification of pediatric ID specialists and eligibility for and content of the board certification examination. After approval from 31 programs, the pediatric ID programs started in 2017 with 8 fellows in 7 programs. The first 6 graduates received board certification in 2020. To date, 61 pediatricians have been board certified as pediatric ID specialists. In parallel, we introduced board certification for pediatricians who work mainly in primary care settings and have a special interest in pediatric ID. This system has certified 338 pediatricians. During and after the development of the programs, we achieved substantial progress in highlighting the pivotal role of pediatric ID specialists, including the establishment and maintenance of antimicrobial stewardship programs, pediatric ID consultations and introduction of viral diagnosis by polymerase chain reaction at institutions. However, several issues need to be addressed, including the establishment of independent pediatric ID departments in institutions, payment of consultation fees, program site visits, maintenance of certification and cultivation of physician-scientists. These challenges will be the focus of future efforts.

Identity conflicts of student affairs officers in a medical university.

INTRODUCTION: Collaboration between student affairs officers and the faculty is important in dealing with the recent rapid changes in medical education, and mutual understanding is essential to ensure that participants become a cohesive social group. This study explores the identity conflicts of student affairs officers in medical universities using the figured worlds theory. METHODS: An exploratory qualitative case study was conducted with 24 student affairs officers at a private medical university in Japan. Data were collected through face-to-face, semi-structured interviews and analysed using thematic analysis from the perspective of a social constructivism paradigm. RESULTS: Qualitative analysis revealed the following three themes regarding the identity conflicts of student affairs officers: differences in the perception of medical students, difficulties in building trusting relationships with the faculty, and resistance to the medical university's traditional atmosphere. Student affairs officers tended to provide support from a student-centred perspective when interacting with medical students, while the faculty employed a teacher-centred perspective. DISCUSSION: To promote understanding between professions, it is necessary to set aside certain professional views and welcome dialogue with other professionals with different values, while also understanding the multi-layered context of medical education, so that conflicts can be handled optimally and relationships can be professionalised for social cohesion.

Transition from undergraduates to residents: A SWOT analysis of the expectations and concerns of Japanese medical graduates during the COVID-19 pandemic.

INTRODUCTION: Interruptions in undergraduate clinical clerkship during the COVID-19 pandemic have reduced the confidence and preparedness of residents beginning their postgraduate training. We explore the thoughts of new residents about this transition and reflect on the support needed. METHODS: An exploratory qualitative case study was conducted with 51 residents. All had experienced interruptions in clinical training due to the pandemic and had just started their postgraduate training. Qualitative data were collected through 6 focus groups and 12 individual follow-up interviews. A thematic analysis was undertaken, and the data were categorised using a Strengths, Weaknesses, Opportunities, and Threats (SWOT) framework. RESULTS: Graduates beginning their residency were aware of their professionalism and independence during the transition. They also faced the predicament of needing close supervision while their supervisors managed pandemic conditions. Residents emphasised the importance of developing relationships with colleagues and supervisors during the transition to residency and wanted direct observation and detailed feedback from their supervisors during procedures. CONCLUSIONS: The experiences of residents were not uniformly negative. In fact, some had developed a positive mindset when entering the clinical field. Medical faculty members reflecting on interactions with new residents and planning future clinical internships could benefit from placing a high value on building relationships among residents, who may expect direct observation and detailed feedback from their supervisors.

Identification of a plasmin-interactive site within the A2 domain of the factor VIII heavy chain.

Factor VIII is activated and inactivated by plasmin by limited proteolysis. In our one-stage clotting assay, these plasmin-catalyzed reactions were inhibited by the addition of isolated factor VIII A2 subunits and by Glu-Gly-Arg-active-site modified factor IXa. SDS-PAGE analysis showed that an anti-A2 monoclonal antibody, recognizing the factor IXa-interactive site (residues 484-509), blocked the plasmin-catalyzed cleavage at Arg(336) and Arg(372) but not at Arg(740). Surface plasmon resonance-based assays and ELISA demonstrated that the A2 subunit bound to active-site modified anhydro-plasmin with high affinity (K(d): 21 nM). Both an anti-A2 monoclonal antibody and a peptide comprising of A2 residues 479-504 blocked A2 binding by approximately 80% and approximately 55%, respectively. Mutant A2 molecules where the basic residues in A2 were converted to alanine were evaluated for binding of anhydro-plasmin. Among the tested mutants, the R484A A2 mutant possessed approximately 250-fold lower affinity than the wild-type A2. The affinities of K377A, K466A, and R471A mutants were decreased by 10-20-fold. The inhibitory effect of R484A mutant on plasmin-catalyzed inactivation of factor VIIIa was approximately 20% of that of wild-type A2. In addition, the inactivation rate by plasmin of factor VIIIa reconstituted with R484A mutant was approximately 3-fold lower than that with wild-type A2. These findings demonstrate that Arg(484) plays a key role within the A2 plasmin-binding site, responsible for plasmin-catalyzed factor VIII(a) inactivation.

Identification of a protein S-interactive site within the A2 domain of the factor VIII heavy chain.

We have recently demonstrated that protein S impairs the intrinsic tenase complex, independent of activated protein C, in competitive interactions between the A2 and A3 domains of factor VIIIa and factor IXa. In the present study, we have identified a protein S-interactive site in the A2 domain of factor VIIIa. Anti-A2 monoclonal antibody recognising a factor IXa-functional region (residues 484-509) on A2, and synthetic peptide inhibited the A2 binding to protein S by approximately 60% and approximately 70%, respectively, in solid-phase binding assays. The 484-509 peptide directly bound to protein S dose-dependently. Covalent cross-linking was observed between the 484-509 peptide and protein S following reaction with EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide). The cross-linked adduct was consistent with 1:1 stoichiometry of reactants. Cross-linking formation was blocked by addition of the 484-497 peptide, but not by the 498-509 peptide. Furthermore, N-terminal sequence analysis of the 484-509 peptide-protein S adduct showed that three sequential residues (S488, R489, and R490) in A2 were not identified, suggesting that these residues participate in cross-link formation. Mutant A2 molecules where these residues were converted to alanine were evaluated for the binding of protein S. The S488A, R489A, and R490A mutants demonstrated approximately four-fold lower affinity than wild-type A2. These results indicate that the 484-509 region in the A2 domain of factor VIIIa, in particular sequential residues at positions 488-490, contributes to a unique protein S-interactive site.

Research during Pediatric Residency Training: A Nationwide Study in Japan.

INTRODUCTION: Training in scholarship is an essential component of postgraduate education. Previous studies worldwide on the research activities of pediatric residents were questionnaires targeting program directors or surveys conducted in a limited number of institutions; however, no nationwide studies have been conducted. The objective of this study was to describe the research activities of pediatric residents. METHODS: We conducted a nationwide cross-sectional study during 2015 and 2016 in Japan. Study data were collected from each resident's logbook submitted to the board examination office and compared by the type of institution, namely, university, children's, or community hospital. RESULTS: Of 1,718 eligible participants, 1,500 participated in this study. Overall, 499 (33.3%) residents trained at national/public university hospitals, 371 (24.7%) at private university hospitals, 140 (9.3%) at children's hospitals, and 490 (32.7%) at community hospitals. Although 1,361 (90.7%) residents gave at least one presentation at an academic conference during their residency, only 235 (15.7%) residents published one or more articles in a peer-reviewed academic medical journal. The proportion of residents who gave at least one presentation (p=0.03) and published at least one study (p<0.01) differed significantly among the types of institutions. Residents at community hospitals gave fewer presentations at conferences (odds ratio [OR] 0.56; 95% confidence interval [95% CI] 0.36-0.87) and published fewer peer-reviewed articles (OR 0.53; 95% CI 0.37-0.76) than residents at national/public university hospitals. CONCLUSIONS: This is apparently the first nationwide study demonstrating that the research activities of pediatric residents consisted mostly of presentations at academic conferences, but also that most residents had not published their research. There was a marked variation in residents' academic activities by institution type.

Protein S down-regulates factor Xase activity independent of activated protein C: specific binding of factor VIII(a) to protein S inhibits interactions with factor IXa.

Protein S functions as an activated protein C (APC)-independent anticoagulant in the inhibition of intrinsic factor X activation, although the precise mechanisms remain to be fully investigated. In the present study, protein S diminished factor VIIIa/factor IXa-dependent factor X activation, independent of APC, in a functional Xa generation assay. The presence of protein S resulted in an c. 17-fold increase in K(m) for factor IXa with factor VIIIa in the factor Xase complex, but an c. twofold decrease in K(m) for factor X. Surface plasmon resonance-based assays showed that factor VIII, particularly the A2 and A3 domains, bound to immobilized protein S (K(d); c. 10 nmol/l). Competition binding assays using Glu-Gly-Arg-active-site modified factor IXa showed that factor IXa inhibited the reaction between protein S and both the A2 and A3 domains. Furthermore, Sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed that the cleavage rate of factor VIIIa at Arg(336) by factor IXa was c. 1.8-fold lower in the presence of protein S than in its absence. These data indicate that protein S not only down-regulates factor VIIIa activity as a cofactor of APC, but also directly impairs the assembly of the factor Xase complex, independent of APC, in a competitive interaction between factor IXa and factor VIIIa.

Anticoagulant activity of M-LAO, L-amino acid oxidase purified from Agkistrodon halys blomhoffii, through selective inhibition of factor IX.

One of haemorrhagic toxins present in snake venoms is L-amino acid oxidase (LAO), which catalyzes the oxidative deamination of L-amino acids with the generation of hydrogen peroxide. Although it is widely accepted that LAO alters platelet function, the effects of LAO on human blood coagulation remain largely unknown. The present study demonstrated, for the first time, that M-LAO, LAO purified from the venom of Agkistrodon halys blomhoffii (Japanese mamushi), possesses an anticoagulant activity. Thrombelastography (TEG) showed that M-LAO significantly delayed the onset and the progress of the coagulation process. In addition, the enzyme prolonged the activated partial thromboplastin time (aPTT) dose-dependently, but had little effect on the prothrombin time (PT), suggesting that its principal activity was mediated in the intrinsic coagulation pathway. Furthermore, M-LAO reduced factor IX procoagulant activity in a dose-dependent manner and did not affect other coagulation factors. These results indicate that M-LAO has an anticoagulant activity that impairs the intrinsic clotting by inhibiting factor IX.

Human factor VIII inhibitor alloantibodies with a C2 epitope inhibit factor Xa-catalyzed factor VIII activation: a new anti-factor VIII inhibitory mechanism.

Factor VIII (FVIII) inhibitor alloantibodies react with the A2, C2, or A3-CI domains of FVIII and inactivate FVIII activity. We recently demonstrated that an anti-C2 monoclonal antibody with a Val2248-Gly2285 epitope, inhibited factor Xa (FXa)-catalyzed FVIII activation, and that a FXa binding site for FVIII was located within residues Thr2253-Gln2270. In this study, we investigated whether anti-C2 alloantibodies inhibit FXa-catalyzed FVIII activation. Anti-C2 alloantibodies from four patients inhibited FVIII activation by FXa in one-stage clotting assay. Furthermore, analysis by SDS-PAGE showed that all alloantibodies inhibited FVIII proteolytic cleavage by FXa independently of phospholipid. To confirm direct inhibition of FVIII and FXa interaction, we examined the effect of alloantibodies on FVIII binding to anhydro-FXa, a catalytically inactive FXa, in ELISA. All alloantibodies and C2-affinity purified F(ab)'2 preparations inhibited FVIII binding to anhydro-FXa dose-dependently. Our results revealed a new inhibitory mechanism of FVIII, mediated by inhibition of FXa in the presence of anti-C2 alloantibodies.

A critical role for thrombin in platelet aggregation under high shear stress.

The serine protease, thrombin, plays a crucial role in both coagulation and platelet activation. Anhydrothrombin (AhT) is a catalytically inactive derivative of thrombin in which dehydroalanine replaces the active-site serine. AhT retains affinity for natural substrates of thrombin and may be a competitive inhibitor of thrombin-mediated coagulation and platelet reactions. In the present study, thrombelastography showed that AhT not only delayed the onset and the progress of the coagulation process but impaired clot strength, indicating that AhT may have both anticoagulant and antiplatelet activity. In addition, AhT prolonged the activated partial thromboplastin time dose-dependently, but had little effect on the prothrombin time, suggesting that its principal activity was mediated in the intrinsic coagulation pathway. AhT inhibited thrombin-induced aggregation of platelet-rich plasma. Complete inhibition of aggregation was evident at a concentration of 1.85 microM AhT. Furthermore, 3.7 microM of AhT almost completely abolished shear-induced platelet aggregation in PRP. Interpretation of this in vitro study requires confirmation in vivo, but the findings suggest that thrombin plays a critical role in shear related platelet mechanisms. AhT may be a useful tool for investigating platelet-based coagulation reactions and may provide the basis for a novel class of antithrombotic agents.

Plasmin-induced procoagulant effects in the blood coagulation: a crucial role of coagulation factors V and VIII.

Plasminogen activators provide effective treatment for patients with acute myocardial infarction. However, paradoxical elevation of thrombin activity associated with failure of clot lysis and recurrent thrombosis has been reported. Generation of thrombin in these circumstances appears to be owing to plasmin (Plm)-induced activation of factor (F) XII. Plm catalyzes proteolysis of several coagulant factors, but the roles of these factors on Plm-mediated procoagulant activity remain to be determined. Recently developed global coagulation assays were used in this investigation. Rotational thromboelastometry using whole blood, clot waveform analysis and thrombin generation tests using plasma, showed that Plm (> or =125 nmol/l) shortened the clotting times in similar dose-dependent manners. In particular, the thrombin generation test, which was unaffected by products of fibrinolysis, revealed the enhanced coagulation with an approximately two-fold increase of peak level of thrombin generation. Studies using alpha2-antiplasmin-deficient plasma revealed that much lower dose of Plm (> or =16 nmol/l) actually contributed to enhancing thrombin generation. The shortening of clotting time could be observed even in the presence of corn trypsin inhibitor, supporting that Plm exerted the procoagulant activity independently of FXII. In addition, using specific coagulation-deficient plasmas, the clot waveform analysis showed that Plm did not shorten the clotting time in only FV-deficient or FVIII-deficient plasma in prothrombin time-based or activated partial thromboplastin time-based assay, respectively. Our results indicated that Plm did possess procoagulant activity in the blood coagulation, and this effect was likely attributed by multicoagulation factors, dependent on FV and/or FVIII.

Determination of a factor VIII-interactive region within plasmin responsible for plasmin-catalysed activation and inactivation of factor VIII(a).

Plasmin, an active form of plasminogen, activates and inactivates factor VIII (FVIII) by limited proteolysis. We have previously identified lysine-binding site-independent plasmin-interactive sites on the FVIII A2 domain responsible for cleavages at Arg336 and Arg372, together with lysine-binding site-dependent plasmin sites on the light chain responsible for cleavage at Lys36. We have now characterised FVIII-interactive regions on plasmin. SDS-PAGE analysis demonstrated that a monoclonal antibody (mAb) against kringle (K)5-catalytic domain (K5-CD) of plasmin significantly blocked plasmin-catalysed cleavages at Arg336 and Arg372. K5-CD fragment and this mAb blocked plasmin-catalysed activation and inactivation of FVIII(a). Anti-K1-2-3 and anti-K4 mAbs blocked plasmin-catalysed cleavages at Lys36, and K1-2-3 and K4 fragments inhibited plasmin-catalysed inactivation of A11-336FVIIIa. The K5-CD preferentially bound to the A2 domain (Kdapp; 52 nM), whilst the K1-2-3 and K4 bound to the light chain (Kdapp; 75 and 106 nM, respectively) in ELISA. Binding was attributed to the A2 484-509 region and A3 1690-1705/1804-1818 region, respectively. 6-aminohexanoic acid, a lysine analogue, significantly inhibited the light chain/K1-2-3 (and K4) binding by approximately 90%, whilst A2/K5-CD binding was moderated by only approximately 35%. Furthermore, an anti-CD antibody blocked plasmin-catalysed cleavage by inhibiting the A2/K5-CD interaction. These data demonstrate that the K5-CD of plasmin (and plasminogen) interacts with the A2 domain independent of lysine-binding site, whilst interactions of K1-2-3 and K4 with the light chain are lysine-binding site-dependent. Interactions between the K5-CD and A2 likely constitute the major regulatory mechanism for activation and inactivation of FVIII(a) mediated by cleavage at Arg372 and Arg336.

The factor VIIIa C2 domain (residues 2228-2240) interacts with the factor IXa Gla domain in the factor Xase complex.

Factor VIIIa functions as a cofactor for factor IXa in the phospholipid surface-dependent activation of factor X. Both the C2 domain of factor VIIIa and the Gla domain of factor IXa are involved in phospholipid binding and are required for the activation of factor X. In this study, we have examined the close relationship between these domains in the factor Xase complex. Enzyme-linked immunosorbent assay-based and surface plasmon resonance-based assays in the absence of phospholipid showed that Glu-Gly-Arg active site-modified factor IXa bound to immobilized recombinant C2 domain (rC2) dose-dependently (Kd = 108 nm). This binding ability was optimal under physiological conditions. A monoclonal antibody against the Gla domain of factor IXa inhibited binding by approximately 95%, and Gla domainless factor IXa failed to bind to rC2. The addition of monoclonal antibody or rC2 with factor VIIIa inhibited factor IXa-catalyzed factor X activation in the absence of phospholipid. Inhibition was not evident, however, in similar experiments in the absence of factor VIIIa, indicating that the C2 domain interacted with the Gla domain of factor IXa. A fragment designated C2-(2182-2259), derived from V8 protease-cleaved rC2, bound to Glu-Gly-Arg active site-modified factor IXa. Competitive assays, using overlapping synthetic peptides encompassing residues 2182-2259, demonstrated that peptide 2228-2240 significantly inhibited both this binding and factor Xa generation, independently of phospholipid. Our results indicated that residues 2228-2240 in the factor VIIIa C2 domain constitutes an interactive site for the Gla domain of factor IXa. The findings provide the first evidence for an essential role for this interaction in factor Xase assembly.

Identification of plasmin-interactive sites in the light chain of factor VIII responsible for proteolytic cleavage at Lys36.

We have recently reported that plasmin likely associates with the factor VIII light chain to proteolyze at Lys36 within the A1 domain. In this study, we determined that the rate of plasmin-catalyzed inactivation on the forms of factor VIIIa containing A1-(1-336) and 1722A3C1C2, reflecting Lys36 cleavage, was reduced by approximately 60%, compared with those containing 1649A3C1C2 and 1690A3C1C2. SDS-PAGE analysis revealed that Lys36 cleavage of factor VIIIa with 1722A3C1C2 was markedly slower than those with 1649A3C1C2 and 1690A3C1C2. Surface plasmon resonance-based assays, using active site-modified anhydro-plasmin (Ah-plasmin) showed that 1722A3C1C2 bound to Ah-plasmin with an approximately 3-fold lower affinity than 1649A3C1C2 or 1690A3C1C2 (Kd, 176, 68.2, and 60.3 nM, respectively). Recombinant A3 bound to Ah-plasmin (Kd, 44.2 nM), whereas C2 failed to bind, confirming the presence of a plasmin-binding site within N terminus of A3. Furthermore, the Glu-Gly-Arg active site-modified factor IXa also blocked 1722A3C1C2 binding to Ah-plasmin by approximately 95%, supporting the presence of another plasmin-binding site overlapping the factor IXa-binding site in A3. In keeping with a major contribution of the lysine-binding sites in plasmin for interaction with the factor VIII light chain, analysis of the A3 sequence revealed two regions involving clustered lysine residues in 1690-1705 and 1804-1818. Two peptides based on these regions blocked 1649A3C1C2 binding to Ah-plasmin by approximately 60% and plasmin-catalyzed Lys36 cleavage of factor VIIIa with A1-(1-336) by approximately 80%. Our findings indicate that an extended surface, centered on residues 1690-1705 and 1804-1818 within the A3 domain, contributes to a unique plasmin-interactive site that promotes plasmin docking during cofactor inactivation by cleavage at Lys36.

Mechanisms of plasmin-catalyzed inactivation of factor VIII: a crucial role for proteolytic cleavage at Arg336 responsible for plasmin-catalyzed factor VIII inactivation.

Plasmin not only functions as a key enzyme in the fibrinolytic system but also directly inactivates factor VIII and other clotting factors such as factor V. However, the mechanisms of plasmin-catalyzed factor VIII inactivation are poorly understood. In this study, levels of factor VIII activity increased approximately 2-fold within 3 min in the presence of plasmin, and subsequently decreased to undetectable levels within 45 min. This time-dependent reaction was not affected by von Willebrand factor and phospholipid. The rate constant of plasmin-catalyzed factor VIIIa inactivation was approximately 12- and approximately 3.7-fold greater than those mediated by factor Xa and activated protein C, respectively. SDS-PAGE analysis showed that plasmin cleaved the heavy chain of factor VIII into two terminal products, A1(37-336) and A2 subunits, by limited proteolysis at Lys(36), Arg(336), Arg(372), and Arg(740). The 80-kDa light chain was converted into a 67-kDa subunit by cleavage at Arg(1689) and Arg(1721), identical to the pattern induced by factor Xa. Plasmin-catalyzed cleavage at Arg(336) proceeded faster than that at Arg(372), in contrast to proteolysis by factor Xa. Furthermore, breakdown was faster than that in the presence of activated protein C, consistent with rapid inactivation of factor VIII. The cleavages at Arg(336) and Lys(36) occurred rapidly in the presence of A2 and A3-C1-C2 subunits, respectively. These results strongly indicated that cleavage at Arg(336) was a central mechanism of plasmin-catalyzed factor VIII inactivation. Furthermore, the cleavages at Arg(336) and Lys(36) appeared to be selectively regulated by the A2 and A3-C1-C2 domains, respectively, interacting with plasmin.