Cryoprecipitate for Alteplase-Related Hemorrhagic Conversion of Acute Ischemic Stroke.

Background: Evidence to support cryoprecipitate for reversal of alteplase-related hemorrhagic conversion of acute ischemic stroke is limited. Guidelines recommend cryoprecipitate as first line treatment, followed by aminocaproic acid as a conditional recommendation with very low-quality evidence. The purpose of this case series was to describe the use of cryoprecipitate for alteplase-related hemorrhagic conversion of acute ischemic stroke. Methods: This was an IRB-approved retrospective case series of adults who received cryoprecipitate for an alteplase-related hemorrhagic conversion of acute ischemic stroke at two comprehensive stroke centers within a large academic medical center. Thromboembolism at 14 days and hemostasis within 24 hours were collected. The outcomes of cryoprecipitate alone vs cryoprecipitate with aminocaproic acid (C + A) were also described. Results: A total of 19 patients were included. Thrombosis occurred in 1/19 (5%) and hemostasis occurred in 4/14 (29%) of evaluable patients. In-hospital mortality was seen in 9/19 (47%) patients. Seventy four percent (14/19) of patients received concomitant blood products other than cryoprecipitate and 63% received a concomitant reversal agent. Thirteen patients received cryoprecipitate alone and six received C + A. Thrombosis was seen in 1/13 (8%) vs 0/6 (0%) and hemostasis occurred in 2/11 (18%) and 2/3 (67%) evaluable cryoprecipitate vs C + A patients respectively. Conclusion: Cryoprecipitate was associated with a low rate of thrombosis and hemostasis for alteplase-associated hemorrhagic conversion of acute ischemic stroke. There was significant heterogeneity in treatment regimens, including the use of and dosing of adjunctive aminocaproic acid and monitoring of fibrinogen levels.

A Low-Dose 4F-PCC Protocol for DOAC-Associated Intracranial Hemorrhage.

PURPOSE: Current guidelines favor 4F-PCC over plasma for reversal of warfarin. Uncertainty remains on the hemostatic effectiveness and thrombotic risk of 4F-PCC for direct-acting oral anticoagulants (DOACs), particularly in patients with intracranial hemorrhage (ICH). This study sought to evaluate the effectiveness and safety of a lower dose protocol of 25 units/kg 4F-PCC for the management of DOAC-associated ICH in a real-world setting. MATERIALS AND METHODS: This was a retrospective study of adult patients who received at least one dose of 4F-PCC from March 2014 to December 2015 for DOAC-associated ICH. The primary outcome was hemostatic effectiveness within 24 hours. The secondary outcome was thromboembolic events within 14 days. RESULTS: Twenty-two patients received 4F-PCC for DOAC-associated ICH and were included in the analysis. Hemostasis was evaluable in 19 patients with post-4F-PCC imaging available and occurred in 18/19 (94.7%) patients. Thromboembolism occurred in 2 out of 22 patients (9.1%). CONCLUSIONS: The use of a lower dose protocol of 25 units/kg of 4F-PCC resulted in high rates of hemostasis in patients with DOAC-associated ICH. Two patients developed thrombotic events within 14 days of 4F-PCC administration.

Aminocaproic Acid for the Reversal of Alteplase: A Case Series.

INTRODUCTION: The evidence to support the use of aminocaproic acid for reversal of alteplase is limited to case reports. Current guidelines recommend cryoprecipitate as first-line treatment, or an antifibrinolytic if cryoprecipitate is unavailable or cannot be used. This case series describes the use of aminocaproic acid for alteplase-related hemorrhage. MATERIALS AND METHODS: Patients who received aminocaproic acid within 48 hours of alteplase from January 1, 2014, to June 30, 2017 were included. Patients were excluded if aminocaproic acid was not administered for an alteplase-related hemorrhage. Thromboembolic complications at 14 days and hemostasis within 24 hours were documented. RESULTS: Sixteen patients received aminocaproic acid for an alteplase-related hemorrhage. Aminocaproic acid was given for hemorrhagic conversion of acute ischemic stroke in 7 of 16 patients and other types of hemorrhages in the remaining patients. Hemostasis occurred in 3 of 10 evaluable patients. Overall mortality was 63% (10/16). Administration of aminocaproic acid varied significantly (50% bolus, 12.5% infusion, 37.5% bolus plus infusion). One of 6 evaluable patients had a thromboembolic event. All patients received concomitant blood products. Cryoprecipitate was administered in 69% (11/16) of patients. CONCLUSIONS: There was no clear relationship between the timing or dose of aminocaproic acid and hemostasis, and it is unclear if administration of aminocaproic acid contributes to hemostasis.

Effectiveness and Tolerability of Conivaptan and Tolvaptan for the Treatment of Hyponatremia in Neurocritically Ill Patients.

STUDY OBJECTIVE: To describe the effectiveness and tolerability of conivaptan and tolvaptan for the correction of hyponatremia in neurocritically ill patients. DESIGN: Retrospective cohort study. SETTING: Neurointensive care units at two academic medical centers. PATIENTS: Thirty-six adults admitted to the neurocritical care unit who received at least one dose of conivaptan (5 patients) or tolvaptan (31 patients) between June 2012 and May 2013. MEASUREMENTS AND MAIN RESULTS: A single oral dose or intravenous bolus was administered to 23 (74%) patients who received tolvaptan and 2 (40%) patients who received conivaptan, respectively. The mean maximal increase in serum sodium level at 24 hours following the last dose compared with baseline was 5.2 mEq/L for conivaptan (p=0.05) and 7.9 mEq/L for tolvaptan (p<0.001). The mean ± SD maximal increases in serum sodium level at 48, 72, and 96 hours following the last dose of vaptan therapy compared with baseline were 5.5 ± 2.2 mEq/L (p=0.01), 5.6 ± 2.0 mEq/L (p=0.005), and 4.8 ± 2.2 mEq/L (p=0.03), respectively. Sodium overcorrection occurred in six patients (19%) receiving tolvaptan and none of the patients receiving conivaptan. Hypotension occurred in 20% of patients receiving conivaptan and 52% of patients receiving tolvaptan, whereas hypokalemia was observed in 40% of patients receiving conivaptan. CONCLUSION: Use of vaptans in neurocritically ill patients led to a significant increase in serum sodium level at 24 hours after the last dose, which was sustained for 96 hours, with the majority of patients receiving a single dose. Risk of sodium overcorrection was high and necessitates appropriate patient selection and frequent monitoring.

Major publications in the critical care pharmacotherapy literature in 2015.

PURPOSE: Recently published practice guidelines and research reports on pharmacotherapy in critical care patient populations are summarized. SUMMARY: The Critical Care Pharmacotherapy Literature Update (CCPLU) Group is composed of over 50 experienced critical care pharmacists who evaluate 31 peer-reviewed journals monthly to identify literature pertaining to pharmacotherapy in critical care populations. Articles are chosen for summarization in a monthly CCPLU Group publication on the basis of applicability and relevance to clinical practice and strength of study design. From January to December 2015, a total of 121 articles were summarized; of these, 3 articles presenting clinical practice guidelines and 12 articles presenting original research findings were objectively selected for inclusion in this review based on their potential to change or reinforce current evidence-based practice. The reviewed guidelines address the management of intracranial hemorrhage (ICH), adult advanced cardiac life support (ACLS) and post-cardiac arrest care, and the management of supraventricular tachycardia (SVT). The reviewed research reports address topics such as nutrition in critically ill adults, administration of ß-lactams for severe sepsis, anticoagulant selection in the context of continuous renal replacement therapy, early goal-directed therapy in septic shock, magnesium use for neuroprotection in acute stroke, and progesterone use in patients with traumatic brain injury. CONCLUSION: Important recent additions to the critical care pharmacy literature include updated joint clinical practice guidelines on the management of spontaneous ICH, ACLS, and SVT.

Treatment of Hyponatremia in Patients with Acute Neurological Injury.

BACKGROUND: Little data exist regarding the practice of sodium management in acute neurologically injured patients. This study describes the practice variations, thresholds for treatment, and effectiveness of treatment in this population. METHODS: This retrospective, multicenter, observational study identified 400 ICU patients, from 17 centers, admitted for ≥48 h with subarachnoid hemorrhage (SAH), traumatic brain injury (TBI), intraparenchymal hemorrhage, or intracranial tumors between January 1, 2011 and July 31, 2012. Data collection included demographics, APACHE II, Glascow Coma Score (GCS), serum sodium (Na+), fluid rate and tonicity, use of sodium-altering therapies, intensive care unit (ICU) and hospital length of stay, and modified Rankin score upon discharge. Data were collected for the first 21 days of ICU admission or ICU discharge, whichever came first. Sodium trigger for treatment defined as the Na+ value prior to treatment with response defined as an increase of ≥4 mEq/L at 24 h. RESULTS: Sodium-altering therapy was initiated in 34 % (137/400) of patients with 23 % (32/137) having Na+ >135 mEq/L at time of treatment initiation. The most common indications for treatment were declining serum Na+ (68/116, 59 %) and cerebral edema with mental status changes (21/116, 18 %). Median Na+ treatment trigger was 133 mEq/L (IQR 129-139) with no difference between diagnoses. Incidence and treatment of hyponatremia was more common in SAH and TBI [SAH (49/106, 46 %), TBI (39/97, 40 %), ICH (27/102, 26 %), tumor (22/95, 23 %); p = 0.001]. The most common initial treatment was hypertonic saline (85/137, 62 %), followed by oral sodium chloride tablets (42/137, 31 %) and fluid restriction (15/137, 11 %). Among treated patients, 60 % had a response at 24 h. Treated patients had lower admission GCS (12 vs. 14, p = 0.02) and higher APACHE II scores (12 vs. 10, p = 0.001). There was no statistically significant difference in outcome when comparing treated and untreated patients. CONCLUSION: Sodium-altering therapy is commonly employed among neurologically injured patients. Hypertonic saline infusions were used first line in more than half of treated patients with the majority having a positive response at 24 h. Further studies are needed to evaluate the impact of various treatments on patient outcomes.

Facilitating the safe use of insulin pens in hospitals through a mentored quality-improvement program.

PURPOSE: Results of the MENTORED QUALITY IMPROVEMENT IMPACT PROGRAM℠ (MQIIP) on Ensuring Insulin Pen Safety in Hospitals, which was part of an ASHP educational initiative aimed at ensuring the safe use of insulin pens in hospitals, are described. METHODS: During this ASHP initiative, which also included continuing-education activities and Web-based resources, distance mentoring by pharmacists with expertise in the safe use of insulin pens was provided to interprofessional teams at 14 hospitals between September 2014 and May 2015. The results of baseline assessments of nursing staff knowledge of insulin pen use, insulin pen storage and labeling audits, and insulin pen injection observations conducted in September and October 2014 were the basis for insulin pen quality-improvement plans. Postintervention data were collected in April and May 2015. RESULTS: Compared with the baseline period, significant improvements in nurses' knowledge of insulin pen use, insulin pen labeling and storage, and insulin pen administration were observed in the postintervention period despite the relatively short time frame for implementation of quality-improvement plans. Program participants are committed to sustaining and building on improvements achieved during the program. The outcome measures described in this report could be adapted by other health systems to identify opportunities to improve the safety of insulin pen use. CONCLUSION: Focused attention on insulin pen safety through an interprofessional team approach during the MQIIP enabled participating sites to detect potential safety issues based on collected data, develop targeted process changes, document improvements, and identify areas requiring further intervention. A sustained organizational commitment is required to ensure the safe use of insulin pen devices in hospitals.

Four-factor prothrombin complex concentrate for life-threatening bleeds or emergent surgery: A retrospective evaluation.

PURPOSE: Previous trials investigating usage of four-factor prothrombin complex concentrate (4F-PCC) excluded patients with various thrombotic risk factors. The objective of this study was to evaluate the safety and effectiveness of 4F-PCC in a real-world setting based on an institutional protocol that does not have strict exclusion criteria. METHODS: This was a retrospective study of adult patients who received 4F-PCC. The primary outcome was a confirmed thromboembolism within 14 days after 4F-PCC administration. Secondary outcomes included international normalized ratio (INR) correction to <1.5 at first draw and incidence of INR rebound for patients undergoing reversal of warfarin and hemostatic effectiveness for patients experiencing a bleed. RESULTS: Ninety-three patients received 4F-PCC. Sixty-three (67.7%) were reversed for bleeding and 30 (32.3%) for surgery. Eleven patients (11.8%) developed a thromboembolism within 14 days. The median (interquartile range) time to event was 5 (2-7) days. Significant risk factors were heparin-induced thrombocytopenia (P= .01) and major surgery within 14 days (P= .02), as well as the presence of >6 thrombotic risk factors (P= .01). For patients post-warfarin reversal, 45/63 (71.4%) achieved INR correction at first draw, 55/63 (87.3%) achieved INR correction within 24 hours, and 14/55 (25.5%) experienced INR rebound. Of these 14 patients, 8 (57.1%) did not receive concomitant vitamin K. CONCLUSIONS: 4F-PCC was associated with a notable thromboembolic risk. All patient-specific risk factors should be considered prior to administration. 4F-PCC remains a useful agent for warfarin reversal. Lack of concomitant vitamin K may contribute to INR rebound.

Is pentobarbital safe and efficacious in the treatment of super-refractory status epilepticus: a cohort study.

INTRODUCTION: Seizures refractory to third-line therapy are also labeled super-refractory status epilepticus (SRSE). These seizures are extremely difficult to control and associated with poor outcome. We aimed to characterize efficacy and side-effects of continuous infusions of pentobarbital (cIV-PTB) treating SRSE. METHODS: We retrospectively reviewed continuous electroencephalography (cEEG) reports for all adults with RSE treated with cIV-PTB between May 1997 and April 2010 at our institution. Patients with post-anoxic SE and those receiving cIV-PTB for reasons other than RSE were excluded. We collected baseline information, cEEG findings, side-effects and functional outcome at discharge and one year. RESULTS: Thirty one SRSE patients treated with cIV-PTB for RSE were identified. Mean age was 48 years old (interquartile range (IQR) 28,63), 26% (N = 8) had a history of epilepsy. Median SE duration was 6.5 days (IQR 4,11) and the mean duration of cIV-PTB was 6 days (IQR 3,14). 74% (N = 23) presented with convulsive SE. Underlying etiology was acute symptomatic seizures in 52% (N = 16; 12/16 with encephalitis), remote 30% (N = 10), and unknown 16% (N = 5). cIV-PTB controlled seizures in 90% (N = 28) of patients but seizures recurred in 48% (N = 15) while weaning cIV-PTB, despite the fact that suppression-burst was attained in 90% (N = 28) of patients and persisted >72 hours in 56% (N = 17). Weaning was successful after adding phenobarbital in 80% (12/15 of the patients with withdrawal seizures). Complications during or after cIV-PTB included pneumonia (32%, N = 10), hypotension requiring pressors (29%, N = 9), urinary tract infection (13%, N = 4), and one patient each with propylene glycol toxicity and cardiac arrest. One-third (35%, N = 11) had no identified new complication after starting cIV-PTB. At one year after discharge, 74% (N = 23) were dead or in a state of unresponsive wakefulness, 16% (N = 5) severely disabled, and 10% (N = 3) had no or minimal disability. Death or unresponsive wakefulness was associated with catastrophic etiology (p = 0.03), but none of the other collected variables. CONCLUSIONS: cIV-PTB effectively aborts SRSE and complications are infrequent; outcome in this highly refractory cohort of patients with devastating underlying etiologies remains poor. Phenobarbital may be particularly helpful when weaning cIV-PTB.

Seizure prophylaxis in neurocritical care: a review of evidence-based support.

Seizures are a well-described complication of acute brain injury and neurosurgery. Antiepileptic drugs (AEDs) are frequently utilized for seizure prophylaxis in neurocritical care patients. In this review, the Neurocritical Care Society Pharmacy Section describes the evidence associated with the use of AEDs for seizure prophylaxis in patients with intracerebral tumors, traumatic brain injury, aneurysmal subarachnoid hemorrhage, craniotomy, ischemic stroke, and intracerebral hemorrhage. Clear evidence indicates that the short-term use of AEDs for seizure prophylaxis in patients with traumatic brain injury and aneurysmal subarachnoid hemorrhage may be beneficial; however, evidence to support the use of AEDs in other disease states is less clear.

Major publications in the critical care pharmacotherapy literature: February 2012 through February 2013.

PURPOSE: Recent impactful additions to the professional literature on the role of pharmacotherapy in treating the critically ill are summarized. SUMMARY: An unusually large number of updated practice guidelines and other publications with broad critical care pharmacotherapy ramifications appeared in the primary biomedical literature during the designated review period (February 2012-February 2013). Hundreds of relevant articles were evaluated by the Critical Care Pharmacotherapy Literature Update group (CCPLU), a national group of pharmacists who routinely monitor 25 peer-reviewed journals for emerging evidence that pertains to rational medication use in the intensive care unit (ICU) setting. From among those articles, 64 were summarized for dissemination to CCPLU members; the 8 publications deemed to have the greatest utility for critical care practitioners, as determined by CCPLU through a voting process, were selected for inclusion in this review, with preference given to evidence meeting high standards of methodological quality. The summaries presented here include (1) important new recommendations on management of pain, agitation, and delirium in critically ill patients, (2) a comprehensive update of a practice guideline issued in 2008 by the Surviving Sepsis Campaign, (3) novel strategies for the prevention and/or treatment of hyperglycemia in critical care, and (4) reports on clinical trials of promising alternative methods of sedation for use in weaning patients from mechanical ventilation. CONCLUSION: This review provides synopses of practice guidelines and other recent additions to the professional literature pertaining to rational medication use in the ICU practice setting.

High-dose midazolam infusion for refractory status epilepticus.

OBJECTIVE: This study compares 2 treatment protocols allowing low vs high continuous IV midazolam (cIV-MDZ) doses. METHODS: We compared adults with refractory status epilepticus treated with a protocol allowing for high-dose cIV-MDZ (n = 100; 2002-2011) with those treated with the previous lower-dose cIV-MDZ (n = 29; 1996-2000). We collected data on baseline characteristics, cIV-MDZ doses, seizure control, hospital course, and outcome. RESULTS: Median maximum cIV-MDZ dose was 0.4 mg/kg/h (interquartile range [IQR] 0.2, 1.0) for the high-dose group and 0.2 mg/kg/h (IQR 0.1, 0.3) for the low-dose group (p < 0.001) with similar duration of infusion. Median time from status epilepticus onset to cIV-MDZ start was 1 day (IQR 1, 3) for the high-dose group and 2 days (IQR 1, 5) for the low-dose group (p = 0.016). "Withdrawal seizures" (occurring within 48 hours of discontinuation of cIV-MDZ) were less frequent in the high-dose group (15% vs 64%, odds ratio 0.10, 95% confidence interval 0.03-0.27). "Ultimate cIV-MDZ failure" (patients requiring change to a different cIV antiepileptic medication) and hospital complications were not different between groups. Hypotension was more frequent with higher cIV-MDZ doses but was not associated with worse outcome. Discharge mortality was lower in the high-dose group (40% vs 62%, odds ratio 0.34, 95% confidence interval 0.13-0.92 in multivariate analysis). CONCLUSIONS: High-dose cIV-MDZ treatment of refractory status epilepticus can be performed safely, is associated with a lower seizure rate after cIV-MDZ discontinuation, and may be associated with lower mortality than traditional lower-dose protocols. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that midazolam at higher infusion rates is associated with a reduction in seizure recurrence within 48 hours after discontinuation and may be associated with lower mortality.

Effectiveness and safety of nicardipine and labetalol infusion for blood pressure management in patients with intracerebral and subarachnoid hemorrhage.

BACKGROUND: Nicardipine and labetalol are two commonly used antihypertensives for treating elevated blood pressures in the setting of intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH). There are no studies comparing these two agents as continuous infusions. METHODS: A retrospective chart review was conducted of patients admitted between November 2009 and January 2011 with ICH and SAH to compare effectiveness and safety between both agents. Percent time spent at goal was set as the primary outcome. The secondary outcomes included blood pressure variability, time to goal, incidence of bradycardia, tachycardia, and hypotension. RESULTS: A total of 81 patients were available for analysis, 10 initiated on labetalol (LAB), 57 on nicardipine (NIC), and 14 required the combination of these agents (COMB) to reach goal. We found no difference between NIC, LAB, and the COMB groups in the median percent time at goal [88 % (61-98); 93 % (51-99); 66 % (25-95), (p = NS)]. Median percentage of blood pressure variability, hypotension, and bradycardia were also comparable between groups, however, more tachycardia was observed in the COMB group versus both LAB and NIC groups (45 vs. 0 vs. 3 %; p < 0.001). Mean time to goal SBP in 24 patients who had BP readings available at 1st h of initiation was 32 ± 34 min in the NIC group and 53 ± 42 min in the LAB group (p = 0.03). CONCLUSIONS: Both agents appear equally effective and safe for blood pressure control in SAH and ICH during the initial admission hours. A prospective study is needed to validate these findings.

Practice variations in the management of status epilepticus.

BACKGROUND: Numerous anticonvulsant agents are now available for treating status epilepticus (SE). However, a paucity of data is available to guide clinicians in the initial treatment of seizures or SE. This study describes the current strategies being employed to treat SE in the U.S.A. METHODS: Fifteen American academic medical centers completed a retrospective, multicenter, observational study by reviewing 10-20 of the most recent cases of SE at their institution prior to December 31, 2009. A multivariate analysis was performed to determine factors associated with cessation of seizures. RESULTS: A total of 150 patients were included. Most patients with SE had a seizure disorder (58%). SE patients required a median of 3 AEDs for treatment. Three quarters of patients received a benzodiazepine as first-line therapy (74.7%). Phenytoin (33.3%) and levetiracetam (10%) were commonly used as the second AED. Continuous infusions of propofol, barbiturate, or benzodiazepine were used in 36% of patients. Median time to resolution of SE was 1 day and was positively associated with presence of a complex partial seizure, AED non-compliance prior to admission, and lorazepam plus another AED as initial therapy. Prolonged ICU length of stay and topiramate therapy prior to admission were negatively associated with SE resolution. Mortality was higher in patients without a history of seizure (22.2 vs. 6.9%, p = 0.006). CONCLUSIONS: The use of a benzodiazepine followed by an AED, such as phenytoin or levetiracetam, is common as first and second-line therapy for SE and appears to be associated with a shorter time to SE resolution. AED selection thereafter is highly variable. Patients without a history of seizure who develop SE had a higher mortality rate.

Prevention of shivering during therapeutic temperature modulation: the Columbia anti-shivering protocol.

BACKGROUND: As the practice of aggressive temperature control has become more commonplace, new clinical problems are arising, of which shivering is the most common. Treatment for shivering while avoiding the negative consequences of many anti-shivering therapies is often difficult. We have developed a stepwise protocol that emphasizes use of the least sedating regimen to achieve adequate shiver control. METHODS: All patients treated with temperature modulating devices in the neurological intensive care unit were prospectively entered into a database. Baseline demographic information, daily temperature goals, best daily GCS, and type and cumulative dose of anti-shivering agents were recorded. RESULTS: We collected 213 patients who underwent 1388 patient days of temperature modulation. Eighty-nine patients underwent hypothermia and 124 patients underwent induced normothermia. In 18% of patients and 33% of the total patient days only none-sedating baseline interventions were needed. The first agent used was most commonly dexmeditomidine at 50% of the time, followed by an opiate and increased doses of propofol. Younger patients, men, and decreased BSA were factors associated with increased number of anti-shivering interventions. CONCLUSIONS: A significant proportion of patients undergoing temperature modulation can be effectively treated for shivering without over-sedation and paralysis. Patients at higher risk for needing more interventions are younger men with decreased BSA.

A targeted UAS-RNAi screen in Drosophila larvae identifies wound closure genes regulating distinct cellular processes.

Robust mechanisms for tissue repair are critical for survival of multicellular organisms. Efficient cutaneous wound repair requires the migration of cells at the wound edge and farther back within the epidermal sheet, but the genes that control and coordinate these migrations remain obscure. This is in part because a systematic screening approach for in vivo identification and classification of postembryonic wound closure genes has yet to be developed. Here, we performed a proof-of-principle reporter-based in vivo RNAi screen in the Drosophila melanogaster larval epidermis to identify genes required for normal wound closure. Among the candidate genes tested were kinases and transcriptional mediators of the Jun N-terminal kinase (JNK) signaling pathway shown to be required for epithelial sheet migration during development. Also targeted were genes involved in actin cytoskeletal remodeling. Importantly, RNAi knockdown of both canonical and noncanonical members of the JNK pathway caused open wounds, as did several genes involved in actin cytoskeletal remodeling. Our analysis of JNK pathway components reveals redundancy among the upstream activating kinases and distinct roles for the downstream transcription factors DJun and DFos. Quantitative and qualitative morphological classification of the open wound phenotypes and evaluation of JNK activation suggest that multiple cellular processes are required in the migrating epidermal cells, including functions specific to cells at the wound edge and others specific to cells farther back within the epidermal sheet. Together, our results identify a new set of conserved wound closure genes, determine putative functional roles for these genes within the migrating epidermal sheet, and provide a template for a broader in vivo RNAi screen to discover the full complement of genes required for wound closure during larval epidermal wound healing.

Fondue and transglutaminase in the Drosophila larval clot.

Hemolymph coagulation is vital for larval hemostasis and important in immunity, yet the molecular basis of coagulation is not well understood in insects. Of the larval clotting factors identified in Drosophila, Fondue is not conserved in other insects, but is notable for its effects on the clot's physical properties, a possible function in the cuticle, and for being a substrate of transglutaminase. Transglutaminase is the only mammalian clotting factor found in Drosophila, and as it acts in coagulation in other invertebrates, it is also likely to be important in clotting in Drosophila. Here we describe a Fondue-GFP fusion construct that labels the cuticle and clot, and show that chemical inhibition and RNAi knockdown of the Drosophila transglutaminase gene affect clot properties and composition in ways similar to knockdown of the fon gene. Thus, Fondue appears to be incorporated into the cuticle and is a key transglutaminase substrate in the clot. This is also the first direct functional confirmation that transglutaminase acts in coagulation in Drosophila.

A role for Hemolectin in coagulation and immunity in Drosophila melanogaster.

Hemolectin has been identified as a candidate clotting factor in Drosophila. We reassessed the domain structure of Hemolectin (Hml) and propose that instead of C-type lectin domains, the two discoidin domains are most likely responsible for the protein's lectin activity. We also tested Hml's role in coagulation and immunity in Drosophila. Here we describe the isolation of a new hml allele in a forward screen for coagulation mutants, and our characterization of this and two other hml alleles, one of which is a functional null. While loss of Hml had strong effects on larval hemolymph coagulation ex vivo, mutant larvae survived wounding. Drosophila thus possesses redundant hemostatic mechanisms. We also found that loss of Hml in immune-handicapped adults rendered them more sensitive to Gram(-) bacterial infection. This demonstrates an immunological role of this clotting protein and reinforces the importance of the clot in insect immunity.

Preliminary characterization of hemolymph coagulation in Anopheles gambiae larvae.

Hemolymph coagulation is a first response to injury, impeding infection, and ending bleeding. Little is known about its molecular basis in insects, but clotting factors have been identified in the fruit fly Drosophila melanogaster. Here, we have begun to study coagulation in the aquatic larvae of the malaria vector mosquito Anopheles gambiae using methods developed for Drosophila. A delicate clot was seen by light microscopy, and pullout and proteomic analysis identified phenoloxidase and apolipophorin-I as major candidate clotting factors. Electron microscopic analysis confirmed clot formation and revealed it contains fine molecular sheets, most likely a result of lipophorin assembly. Phenoloxidase appears to be more critical in clot formation in Anopheles than in Drosophila. The Anopheles larval clot thus differs in formation, structure, and composition from the clot in Drosophila, confirming the need to study coagulation in different insect species to learn more about its evolution and adaptation to different lifestyles.